Molecular Epidemiology of Human Enterovirus 71 in the United Kingdom from 1998 to 2006

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2008, p Vol. 46, No /08/$ doi: /jcm Copyright 2008, American Society for Microbiology. All Rights Reserved. Molecular Epidemiology of Human Enterovirus 71 in the United Kingdom from 1998 to 2006 Jon M. Bible, 1 Miren Iturriza-Gomara, 2 Brian Megson, 2 David Brown, 2 Panagiotis Pantelidis, 3 Pam Earl, 4 Justin Bendig, 4 and C. Y. William Tong 1,5 * Infection and Immunology Delivery Unit, Guy s and St Thomas NHS Foundation Trust, London, United Kingdom 1 ; Enteric Virus Unit, Virus Reference Department, Centre for Infections, Health Protection Agency, London, United Kingdom 2 ; Interstitial Lung Disease Group, Department of Population Genetics and Gene Therapy, National Heart and Lung Institute, Imperial College, School of Medicine, London, United Kingdom 3 ; Department of Medical Microbiology, Epsom and St. Helier University Hospitals, Epsom, Surrey, United Kingdom 4 ; and Department of Infectious Diseases, King s College London School of Medicine, London, United Kingdom 5 Received 2 April 2008/Returned for modification 9 June 2008/Accepted 11 July 2008 The last decade witnessed a significant increase in epidemic activity of human enterovirus 71 (EV71) in the Western Pacific Region (WPR). In most European countries, this risk is unrecognized despite occasional cases of severe disease and two severe outbreaks in Eastern Europe 30 years ago. In this study we report the first examination of the molecular epidemiology of EV71 in the United Kingdom from 1998 to Genomic regions encoding the 1D coat protein (VP1) and 3D polymerase (Pol) from 32 EV71 isolates associated with neurological or cutaneous manifestations were sequenced. Phylogenetic analyses of VP1 and 3D Pol sequences identified genotype C as the dominant strain. Several United Kingdom isolates had genetic linkages with predated C1 or C2 strains from Europe and the WPR. Recombination events were not detected between United Kingdom strains. However, a previously published Taiwanese strain was identified as an intergenotypic recombinant. EV71 genotype C appears to have continuous circulation in the United Kingdom from 1998 to 2006 with repeated introductions of new strains replacing previous strains. It is necessary to continuously monitor the molecular evolution and recombination events of EV71. In the last 10 years human enterovirus 71 (EV71), a species A enterovirus, has emerged as a significant nonpolio enterovirus (NPEV) with potential to cause serious neurological disease sometimes in large outbreaks. Although most EV71 infections commonly result in self-limiting conditions such as hand, foot, and mouth disease (HFMD), herpangina or aseptic meningitis, some cases are associated with severe neurological complications, such as encephalitis and poliomyelitis-like paralysis (17). The rapid development of EV71-induced brainstem encephalitis generally carries a poor prognosis with high case fatality rates (12, 25, 28), although immunopathology may also be involved in the pathogenesis (13). In contrast, coxsackievirus A16 (CVA-16), another closely related species A enterovirus and major causative agent of HFMD, is rarely associated with neurological conditions and death. Phylogenetic analyses of EV71 1D coat protein (VP1) have demonstrated the development of three independent genetic lineages, genotypes A, B, and C (1), which can be further subdivided into genogroups: B1 to B5 and C1 to C5 (2, 14, 15, 18). Outbreaks of EV71 infection have appeared in a global series originating in the United States in 1969, going through Europe, and moving into countries constituting the western pacific region (WPR): Australia, Taiwan, Malaysia, Singapore, China, Hong Kong, and Japan (12, 13, 21, 23, 24, 25). In the * Corresponding author. Mailing address: Infection and Immunology Unit, Guys and St Thomas NHS Foundation Trust, 5th Floor, North Wing, St Thomas Hospital, London SE1 7EH, United Kingdom. Phone: Fax: Published ahead of print on 23 July WPR, some of the world s major HFMD epidemics, with large numbers of neurology-associated fatalities, have occurred (6, 7, 9, 29). Frequent mutation constitutes a major part in generating EV71 genetic diversity throughout its evolution over the past decades. Moreover, the cocirculation of mixed EV71 populations could favor recombination and further contribute to strain diversity (11). It is evident from phylogenetic analysis that two of the major epidemics in 1990s, namely, Malaysia in 1997 and Taiwan in 1998, were caused by quite different prevalent strains belonging to genogroups B3 and C2, respectively (22, 23, 29). Indeed, four distinct EV71 genogroups C2, B4, C4, and B5 were found in succession to be responsible for outbreaks of HFMD in Japan over a 6-year period (15). It is therefore clear that a diverse population of pathogenic EV71 strains circulate widely and that temporal change in the relative prevalence of particular genogroups of EV71 is possible. There is considerable awareness among public health authorities on the emergence of EV71 in WPR countries, which has led to the formation of the Asia-Pacific Enterovirus Surveillance Network (APNET; Similarly, collaboration was initiated in 2005 between the National Commission for Polio Eradication and National Reference Centre for Poliomyelitis and Enteroviruses to monitor NPEV and polioviruses in Germany (Robert Koch Institute) (20). However, the recognition of risk potential is not mirrored in many other European countries, including the United Kingdom. Sequence data covering the complete genomes of EV71 are available for 22 representatives of the EV71 serotype in genetic databases with only one representative isolate from Europe (30). 3192

2 VOL. 46, 2008 MOLECULAR EPIDEMIOLOGY OF HUMAN EV Historically, the most severe European EV71 outbreaks recorded occurred in Bulgaria in 1975 (4) and in Hungary in 1978 (3). Sporadic cases with neurological features were reported in France in 1979 (31), Germany in 1998 (8), and Cyprus between 2000 and 2002 (19). More recently, a study identified high percentages of asymptomatic EV71 infection from a wide geographic distribution in Norway (30). However, there has been no specific study undertaken to ascertain the epidemiology and molecular evolution of EV71 within the United Kingdom. We report here the complete coding sequences of the structural protein VP1 and the nonstructural 3D polymerase (Pol) from 32 United Kingdom EV71 isolates obtained from clinical specimens collected between 1998 and MATERIALS AND METHODS Specimens and virus isolation. A total of 32 EV71 isolates associated with neurological complications and/or cutaneous manifestations were studied. All specimens were from United Kingdom patients collected over the period from 1998 to 2006 from the two national enterovirus reference laboratories: Epsom Specialist Virology Unit, West Park Hospital, Epsom, Surrey, United Kingdom (n 19, 1998 to 2004) and Enteric Virus Unit, Virus Reference Department, Centre for Infections, Health Protection Agency, London, United Kingdom (n 12, 2005 to 2006). An additional EV71 isolate came from a single fatal case (in 2006) from the Department of Infection, Guy s and St Thomas NHS foundation Trust. Human embryonic lung fibroblasts and human liver hepatoma cell line (PLC) were used for virus isolation. RNA extraction and RT. Total cellular RNA was extracted from 140 l of cell culture supernatants by using a QIAamp Viral RNA spin column (Qiagen, Hilden, Germany) according to the manufacturer s instructions. Two reverse transcription (RT) strategies were used for production of viral cdna: a random hexamer primer approach using Moloney murine leukemia virus and a genespecific (GS) primer approach using Superscript III (Invitrogen, Paisley, United Kingdom). The random primer RT reaction consisted of, 2 l of RNA, 40 U of RNase inhibitor, 200 U of Moloney murine leukemia virus (Invitrogen), 0.3 lof hexamer (300 ng/ l; Invitrogen), 6 l of5 first-strand reaction buffer, and 200 M concentrations of each deoxynucleoside triphosphate in a total volume of 30 l. cdna synthesis was performed at 25 C for 10 min, 37 C for 60 min, and 94 C for 5 min. For GS RT, 2 l of RNA template, 2 M primer, and 10 mm deoxynucleoside triphosphates were denatured at 65 C 5 min and chilled on ice before the addition of 200 U of Superscript III, 40 U of RNasin (Invitrogen), 0.1 M dithiothreitol, and 4 l of5 first-strand reaction buffer. A reaction temperature of 50 C was optimal for cdna synthesis, followed by 5 min at 70 C for enzyme inactivation. PCR amplification and sequencing. Primers used for amplification of VP1 and 3D pol regions are listed in Table 1. Two sets of overlapping degenerate primer pairs were designed for each region. Primer pair 3DF/3UTRGS amplified optimally at 50 C using a GS RT product for priming. All PCRs were carried out with 2 l of the RT product, 12.5 l of Hotstart Taq Mastermix (Qiagen), and 2.5 l of each primers (10 M) in a total volume of 25 l. After initial denaturation and activation of Hotstart Pol at 95 C for 15 min, there were 40 cycles of amplification consisting of 95 C for 30 s, followed by annealing at 48 C for 30 s and 72 C for 60 s, with a final extension of 72 C for 5 min. Correctly sized PCR products were directly sequenced bidirectionally by using BigDye 3.1 chemistry on an ABI 3730XL sequencer (Applied Biosystems, Warrington, United Kingdom). The sequencing reactions were performed with the same amplification primers as used in the PCR. Sequence assembly and phylogenetic analysis. Sequence data for each isolate was formatted and compiled into contiguous segments by using Editseq and Seqman software (DNASTAR, Madison, WI). Nucleotide sequences of the United Kingdom strains in the genomic regions VP1 (891 bp) and 3D Pol (1,391 bp) were compared to those of the prototype and others isolated elsewhere in the world. Nucleotide sequences were aligned by using CLUSTAL W software (version 1.8.3; Bioinformatics Center, Kyoto University, Japan [ (26) and CLUSTALX software (version 1.8.1; Plate-Forme de Bio-Informatique, France [ (27). For phylogenetic analysis of VP1 gene sequences, United Kingdom EV71 VP1s from the present study and 79 VP1s from EV71 strains available in the GenBank database ( were used, displayed as a dendrogram containing 111 strains and CVA-16 as an outgroup (Fig. TABLE 1. Oligonucleotide primers used for characterization of United Kingdom isolates a Primer Position Sequence Usage VP3F ARTCHACMWTRYTRG GYCAGT VP1R AYCCRTCRTAAAACC AYTGRTADGC 1DF GANTCYAGRGAMTCH YTNGCWTGGC 2BR CYGTSARGGTRACCAT RTCRTARTC 3CF ACYATYTGGGTDGAR CAYAAAYT 3DR RTCATTYARCTRVGC YTCWAT 3DF SYARRATGAARTYHT AYATGGA 3UTRGS TTTTTTTTTTTTGCT ATTC 1). A similar approach was used for the 3D Pol sequences. Phylogenetic trees were constructed by neighbor-joining using the Kimura two-parameter distance method in the PHYLIP Phylogeny Inference Package (version 3.6; Department of Genome Sciences, University of Washington, Seattle [ (6) and plotted by using TreeView software (version 1.6.6; Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland [ (17). Bootstrap analysis with 1,000 pseudo-replicates provided an estimate of reliability for phylogenetic reconstructions. SimPlot program (version 3.5.1; [ (10), with a sliding window of 200 nucleotides in 20 nucleotide steps was used to determine the location of recombination events. Nucleotide sequence accession numbers. EV71 strains have been submitted to the European Molecular Biology Laboratory database ( /embl) under accession numbers AM to AM for VP1 sequences and accession numbers AM to AM for 3D Pol sequences. RESULTS b RT- a Primers were designed using Oligo 4.0 (Molecular Biology Insights, Inc.). N A, C, G, T; V G, A, C; D G, A, T; H A, T, C; W A, T; M A, C; R A, G; S G, C; Y C, T. Primer positions are relative to the genome of genotype C EV71 isolate 2086/98. b SEQ, sequencing. Clinical features and EV71 genotype. Clinical features recorded for the patients, including the genotypes of the isolates, are presented in Table 2. Notably, six cases were HFMD, all genogroup C1. Ten had nonspecific symptoms of fever, rash, irritability, diarrhea, vomiting, or respiratory symptoms (eight C1; two C2). Thirteen had neurological related symptoms: nine had meningitis (six C1; three C2), one had fatal encephalitis (C2), one had walking difficulty (C2), and two had unspecified neurological diseases (C1). No clinical data were available for three patients (C1). Genetic divergence of VP1 and 3D Pol regions. Pairwise nucleotide and amino acid comparisons of the individual regions showed variability to be considerable over the time period. The VP1 gene sequenced here (nucleotides 2443 to 3334 relative to the prototypic sequence BrCr) showed an identity of 88.7% at the nucleotide level and 98.6% identity in protein between the United Kingdom isolates. When United Kingdom sequences and 79 sequences from GenBank were compared, a total of 79 coding changes were found at 61 different positions across the VP1 capsid protein. In addition, nucleotide sequence identity for the 3D Pol region (nucleotides 5937 to

3 3194 BIBLE ET AL. J. CLIN. MICROBIOL. FIG. 1. Phylogenetic relationship among a total of 111 worldwide EV71 strains based on alignment of complete VP1 coding sequences. Thirty-two United Kingdom strains are from the present study, and seventy-nine are from GenBank. United Kingdom strains are highlighted in bold-face. Trees were constructed by the neighbor-joining 7322) between United Kingdom strains was in the range 88.4 to 99.9%, while the amino acid identity was between 97.4 and 100%. Comparison of 22 GenBank sequences with United Kingdom sequences identified 128 amino acid changes over 105 positions in the 3D Pol coding region. Phylogenetic analysis of VP1 coding region. The phylogenetic relationship among 111 worldwide EV71 strains (Table 2), including the single prototype isolate BrCr, was inferred by neighbor-joining based on complete VP1 coding regions. The VP1 of the reference CVA-16 strain (G-10) was included in the analysis as an outgroup. Using these methods, the majority of EV71 isolates (25 of 32) circulating within the United Kingdom were identified as C1 genogroup strains. The remaining seven were identified as genogroup C2. All published EV71 VP1 sequences clustered into the three distinct genotypes A, B, and C in our phylogenetic analysis. However, in the presence of intergenotypic recombinant TW/ 2272/98, our analysis shows genotype A strain BrCr segregating within the genotype B phylogenetic branch (Fig. 1 and 2). Overall, the United Kingdom EV71 strains were closely related in both C1 and C2 genogroups but also showed relatedness to precedent isolated strains from other countries. Three main clusters within the C1 genogroup are evident on the dendrogram (Fig. 1). The first C1 cluster (bootstrap value of 92.1%) supports previously reported phylogenetic relationships (1) between an early Australian C1 isolate (2623-AUS- 86) and other isolates spread though North America during 1988 to Another branch in the phylogenetic tree separates the sequences into two further large C1 clusters (bootstrap value of 91.8%). One of the clusters shows a genetic linkage between 6 United Kingdom EV71 isolates from 1998 to 2001 and 18 GenBank strains isolated from two outbreaks in Malaysia (0749-MAA-99, 0756-MAA-97, 0808-MAA-98, 0557-MAA-98, 0283-MAA-97, 0113-MAA-00, 0807-MAA-00, 0832-MAA-00, 0836-MAA-00, 0838-MAA-99, 0915-MAA-00, 0948-MAA-00, 0389-MAA-00, 0937-MAA-00, 0774-MAA-00, and MAA-00) and Singapore (4575/SIN/98) between 1997 and A single German isolate from 2003 (Germany/356/2003) is also included in this branch. Overall, nucleotide identities for this group are high; the values range between 96.6 and 100%. The third C1 cluster contains 19 sequences from United Kingdom isolates, which forms two sublineages with a lower bootstrap value of 55.8%. This sublineage separation might be explained by temporal differences between the isolates in that 8 United Kingdom strains in one sublineage came from the period from 2005 to 2006, whereas the remaining 11 strains in another sublineage were from 2001 to A Norwegian C1 sequence from 2003 (804/NO/03) was found to cluster with the former group, whereas a German C1 strain from 2003 (Germethod using PHYLIP (5) and plotted by using TreeView (16). Bootstrap values of 1,000 replicates for major lineages are displayed as numbers at the nodes. CVA-16 (G-10) was used as an outgroup. The corresponding VP1 genotype and genogroup of each cluster are indicated by vertical bars. The EV71 prototype strain BrCr is the only example of genotype A, which in the absence of the intergenotypic recombinant TW/2272/98 forms an independent phylogenetic branch (data not shown).

4 VOL. 46, 2008 MOLECULAR EPIDEMIOLOGY OF HUMAN EV TABLE 2. EV71 strains used in the construction of phylogenetic trees Isolate Yr Origin Clinical details a Genogroup GenBank no AUS Australia Encephalitis C2 AF AUS Australia NA C2 AF F/AUS/6/ Australia Meningitis C2 AF M/AUS/2/ Australia HFMD C2 AF M/AUS/5/ Australia Meningitis C2 AF M/AUS/3/ Australia Myelitis C2 AF F/AUS/ Australia Severe neurological C2 DQ AUS Australia NA C1 AF M/AUS/12/ Australia HFMD C1 AF /AUS/ Australia HFMD C1 AF CAN Canada NA C1 AF SHZH China NA C4 AY SHZH China NA C4 AF Germany/356/ Germany NA C1 AY Germany/802/ Germany NA C1 AY /Yamagata/ Japan HFMD C2 AB /Yamagata/ Japan Herpangia C2 AB /Yamagata/ Japan HFMD C2 AB /Yamagata/ Japan HFMD C2 AB yamagata Japan HFMD C2 AB yamagata Japan HFMD C2 AB yamagata Japan HFMD C2 AB yamagata Japan HFMD C2 AB MAA Malaysia HFMD, meningitis C1 AY MAA Malaysia NA C1 AF MAA Malaysia Meningitis C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia HFMD C1 AY MAA Malaysia Ulcer swab C1 AY MAA Malaysia HFMD C1 AY EV71/SARSHA Malaysia HFMD B3 AM UH1/PM/ Malaysia Fatal B4 AM EV71/9/97/SHA Malaysia Fatal B4 AJ EV71/SAR/SHA Malaysia HFMD B4 AM ENT/PM/SHA Malaysia HFMD C2 AM ENT/PM/SHA Malaysia HFMD C2 AM NM New Mexico NA C1 AF /NO/ Norway Asymptomatic C1 DQ CVA-16 (G-10) 1951 SAF HFMD N/A U /SIN/ Singapore HFMD C1 AF /SIN/ Singapore Fatal B4 AF /SIN/ Singapore Nonfatal B4 AF N5055-TW Taiwan Encephalitis C2 AY N5385-TW Taiwan HFMD C2 AY H0139-TW Taiwan Encephalitis C2 AY N4643-TW Taiwan Fatal C2 AY N5811-TW Taiwan HFMD C2 AY / Taiwan HFMD C2 AF TW/2272/ Taiwan Fatal C/B AF PINF754A 1998 Taiwan NA C2 DQ / Taiwan HFMD C2 AF / Taiwan HFMD C2 AF NCKU Taiwan Fatal C2 AF / Taiwan Fatal C2 AF A/ Taiwan HFMD C2 AF EP/17728/ United Kingdom Tonsilitis C1 AM EP/13372/ United Kingdom Aseptic meningitis C1 AM EP/9027/ United Kingdom HFMD C1 AM EP/5622/ United Kingdom Aseptic meningitis C2 AM Continued on following page

5 3196 BIBLE ET AL. J. CLIN. MICROBIOL. TABLE 2 Continued Isolate Yr Origin Clinical details a Genogroup GenBank no. EP/7414/ United Kingdom Bronchiolitis C2 AM EP/449/ United Kingdom V&D C1 AM EP/11168/ United Kingdom HFMD C1 AM EP/2063/ United Kingdom HFMD C1 AM EP/2405/ United Kingdom HFMD C1 AM EP/4709/ United Kingdom Meningitis C1 AM EP/5746/ United Kingdom Meningitis C1 AM EP/8277/ United Kingdom NA C1 AM EP/11052/ United Kingdom NA C1 AM EP/12105/ United Kingdom Fever, rash, cough C1 AM EP/12081/ United Kingdom HFMD C1 AM EP/2353/ United Kingdom NA C1 AM EP/3267/ United Kingdom URTI, rash C1 AM EP/2826/ United Kingdom Meningitis C1 AM EP/8300/ United Kingdom HFMD C1 AM H United Kingdom Pyrexia, neurological C1 AM H United Kingdom Neurological C1 AM H United Kingdom Meningitis C1 AM H United Kingdom Pyrexia, irritable C1 AM H United Kingdom Meningitis C1 AM H United Kingdom Pyrexia C1 AM H United Kingdom Sepsis, V&D C1 AM H United Kingdom Pyrexia, irritable C1 AM H United Kingdom Meningitis C2 AM H United Kingdom Irritable, rash C2 AM H United Kingdom Pyrexia, meningitis C2 AM H United Kingdom Difficulty walking C2 AM STH/MCN/ United Kingdom Panencephalitis, fatal C2 AM BrCr 1970 United States Aseptic meningitis A U TX United States NA C2 AF MS/7423/ United States AFP B2 U TX United States NA C1 AF NC United States NA C1 AF VA United States NA C1 AF CA United States Paralysis C1 AF TX United States NA C1 AF TX United States NA C1 AF CA United States Meningitis C1 AF OK United States NA C1 AF /0R/ United States NA C1 AF TX United States NA C1 AF CA United States Meningitis C1 AF WA United States NA C1 AF VA United States NA C1 AF a AFP, acute flaccid paralysis; NA, not available; URTI, upper respiratory tract infection; V&D, vomiting and diarrhea. many/802/2003) and a non-european C1 isolate (1M/AUS/12/ 00) originating from an HFMD case in Australia in 2000 were found to cluster with the latter group. Overall nucleotide identities were very high: 99.6 to 100% in the 2005-to-2006 group and 98.2 to 99.6% in the 2001-to-2004 group. The VP1 constructed dendrogram has good bootstrap support for the grouping of United Kingdom and GenBank isolates into three evolutionary branches within genogroup C2. The first branch (bootstrap value of 99.9%) consists of strains from four cutaneous cases (1116/Yamagata/00, 1814/Yamagata/01, 1695/Yamagata/01, and 1585/Yamagata/00) in Japan (2000 to 2001). The apparent sequence separateness of this branch is in concordance with the original article (5), which showed all other Japanese C2 strains in the study to have less than 95% nucleotide identity with these particular four isolates. The second C2 cluster in the phylogenetic tree is entirely composed of viruses isolated during the 1998 EV71 epidemic in Taiwan (N5811-TW-98, N5385-TW-98, H0139-TW-98, N4643-TW-98, N5055-TW-98, 5746/98, PINF754A, 2086/98, 1245A/98, NCKU9822, 6092/98, and 4643/98). The third C2 cluster includes seven United Kingdom C2 strains that show genetic relatedness (93.1 to 97.6% sequence identity) to EV71 that originated in Australia in 1995 (2642-AUS-95 and AUS-95) and circulated in the United States (2286-TX-97) and Malaysia (ENT/PM/SHA52, ENT/PM/SHA71) in 1997 and in Japan in 1998 (671/Yamagata/98, 705/Yamagata/98, 957/ Yamagata/98, and 932/Yamagata/98). Two United Kingdom strains from 1999 (EP/5622/99 and EP/7414/99) show highest sequence identity (96.6 to 99.8%) with five Australian strains (7F/AUS/6/99, 27M/AUS/2/99, 2M/AUS/3/99, 5M/AUS/5/99, and 6F/AUS/99) originating from an epidemic in western Australia in the same year (14). The five remaining United Kingdom C2 isolates from 2006 form a close cluster with no related strains identified from elsewhere.

6 VOL. 46, 2008 MOLECULAR EPIDEMIOLOGY OF HUMAN EV FIG. 2. Dendrograms showing the genetic relationships of 54 EV71 isolates based on an alignment of the complete VP1 and 3D Pol genomic sequences. Thirty-two United Kingdom strains are from the present study, and twenty-two are from GenBank. United Kingdom strains are highlighted in boldface. Trees were constructed by neighbor-joining method by using PHYLIP (5) and plotted by using TreeView (16). Bootstrap values of 1,000 replicates for major lineages are displayed as numbers at the nodes. CVA-16 (G-10) was used as an outgroup. The corresponding VP1 genotype and genogroup of each cluster are indicated by vertical bars. GenBank EV71 strain (TW/2272/98) isolated from a fatal case in Taiwan 1998 grouped as a genotype B based on VP1 analysis (asterisk) but was subsequently repositioned as a genogroup C2 when tested in the 3D Pol analysis. The EV71 prototype strain BrCr is the only example of genotype A, which in the absence of the intergenotypic recombinant TW/2272/98 forms an independent phylogenetic branch (data not shown). Phylogenetic analysis of 3D Pol coding region. To further examine the robustness of strain relationships and assess whether recombination had occurred among United Kingdom and global isolates, RT-PCR products for the 3D Pol coding region from 32 United Kingdom strains were sequenced and compared to 22 3D Pol sequences obtained from HEV71 genomic sequences in GenBank. The 3D Pol of the reference CVA-16 strain G-10 was included in the phylogenetic analysis as an outgroup. A VP1 tree was also constructed using identical sequences to allow direct comparability of phylogenies based on structural versus nonstructural regions. In general, the shape and grouping of strains in the 3D Pol and VP1 constructed trees are consistent with each other; however, some strains exhibit a shift in branch position (Fig. 2, asterisk). In particular, the Norwegian C1 isolate, 804/NO/03, appears intermediate between two United Kingdom C1 clusters in the 3D Pol analysis, whereas VP1 analysis places it closer to the eight United Kingdom strains from 2005 to United Kingdom isolate EP/2063/01, along with EP/4709/01 and EP/5746/98, appeared phylogenetically closer in the 3D Pol region and segregate differently compared to VP1 analysis, although this did not receive high bootstrap support (60%). It is possible that these regroupings may reflect minor localized genetic changes in certain regions of the viral genome. Both 3D Pol and VP1 phylogenetic trees produced two strongly supported clusters concerning the C2 genogroup viruses. However, there is a difference in clustering by 3D Pol analysis relating to the more recent United Kingdom strains, which are grouped on a separate branch. However, both trees indicate that the strains causing the epidemic in Western Australia in 1999 (14) were similar to the EV71 C2 isolates in the United Kingdom in the same year, indicating transcontinental transmission. Finally, a major incongruence was noted between the 3D Pol and VP1 constructed trees. However, this did not involve any United Kingdom strains. In this analysis the GenBank EV71 strain (TW/2272/98) isolated from a fatal case in Taiwan 1998 was grouped as a genotype B virus based on VP1 analysis (Fig. 2, asterisk) but subsequently repositioned to genogroup C2 in the 3D Pol analysis. To examine whether recombination had occurred, aligned full-length EV71 genomes, including the C2 genogroup prototype NCKU9822, TW/2272/98, 2086/98, and B

7 3198 BIBLE ET AL. J. CLIN. MICROBIOL. FIG. 3. Similarity analysis of complete EV71 genomes calculated by SimPlot (10) using a sliding window of 200 nucleotides moving in 20-bp steps. All positions with gaps were deleted, and the similarity was plotted by using the Kimura two-parameter distance model. Each point represents the similarity between the query sequence and a given heterologous sequence. The approximate position within the EV71 genome is indicated above the similarity analysis. The plot shows a comparison of strains 2086/98, TW/2272/98, and MS/7423/87 versus NCKU9822. genotype reference sequence (MS/7423/87), were included in a similarity analysis using SimPlot (10) (Fig. 3). It is evident from the similarity plot that identity is high ( 95%) between the C2 genogroup TW/2272/98, 2086/98, and NCKU9822 isolates over the 5 untranslated region and coat proteins VP4, VP2, and VP3. However, between nucleotides 2440 and 3300 (which encode the VP1), sequence identity between TW/ 2272/98 and the other C2 genogroup sequences is much lower ( 75%), displaying a shift toward the B genotype sequence (MS/7423/87). This analysis supports the observation that the two regions used in the dendrogram reconstruction display different evolutionary histories, indicating that isolate TW/ 2272/98 is a result of intergenotypic recombination. DISCUSSION Major epidemics across the world (6, 7, 9, 29) have seen EV71 becoming acknowledged as a significant human pathogen. However, little is known about its epidemiology in the United Kingdom. In the present study we present the first molecular epidemiological analysis of EV71 strains isolated in the United Kingdom over a period of 8 years ending in The virus strains studied were isolates collected from two national enterovirus reference laboratories that received enterovirus-positive specimens from all over the country for typing and further characterization. Similar to the experience in other countries, EV71 infection in the United Kingdom was associated with cases of mild illness, including HFMD, with a subset of cases developing neurological symptoms, including one fatality. Using phylogenetic analysis of 32 EV71 VP1 capsid coding sequences, genotype C has been identified as the sole genotype circulating in the United Kingdom over the past 8 years. It is possible that certain EV71 genogroups may be more adaptable to grow in cell culture and that this may lead to bias. However, characterization of an additional 14 EV71 strains that failed to grow in cell culture directly from the clinical samples using partial VP1 sequencing also identified them as genotype C (data not shown). The parental cell line may also have dictated the presence of particular nucleotide changes within the viral genomes; however, this seems unlikely since the 19 United Kingdom strains cultivated in HeLa cell lines are distributed between two genetically separated C1 clusters (bootstrapped value of 91.8%). In addition, the highest nucleotide sequence identities in the study (99.6 to 100%) occur for United Kingdom strains cultivated in PLC cell lines compared to a Norwegian strain (804/NO/03) isolated directly from a stool sample, suggesting the cell line had a negligible influence on the native sequence of the VP1 region for these United Kingdom strains. Continuous circulation of EV71 in the community was evident since, apart from the year 2003, EV71 was isolated every year from 1998 to 2006 (Table 2). Of the isolates, genogroup C1 was identified as the most prevalent (78%), while C2 was present in 12% of cases. The genetic variation observed within the genotype was consistent with other studies, with more than 88.7% identity at the nucleotide level and an amino acid identity of at least 98.6% (1). Previous studies have suggested that genogroup C1 is composed of two separate lineages (14); however, these analyses have been dominated by isolates from WPR outbreaks until now. Our analysis of evolutionary relationships between 32 United Kingdom strains and 79 others from across the world indicates genogroup C1 may split into three lineages, each with a bootstrap value greater than 90% (Fig. 1). Consistent with other studies, earlier strains of genotype C originating in Australia in 1986 that spread through small outbreaks in the United States in the 1990s were grouped together to comprise the first C1 lineage. The second C1 lineage contains United Kingdom strains from 1998 to 2001, which were closely related to strains from Malaysia (isolated in 1997) and Singapore (isolated in 1998). Between 1997

8 VOL. 46, 2008 MOLECULAR EPIDEMIOLOGY OF HUMAN EV and 2000 Malaysia experienced endemic circulation of four distinct EV71 genogroups (B3, B4, C1, and C2), of which C1 and B4 became more prevalent resulting in the 2000 epidemic (6). Our findings suggest that this lineage of genogroup C1 viruses have circulated globally rather than being confined to Northern Asia (2, 6, 14, 23). Interestingly, a similar strain was isolated in Germany in 2003 (Germany/356/2003), indicating further spread to continental Europe. The third C1 lineage appears to branch into two smaller sublineages. Examples of temporally and geographically separated strains predating the United Kingdom isolates were found in each sublineage. Of the 11 United Kingdom strains dating from 2001 to 2004, some are almost genetically identical (i.e., nucleotide identities of 98.3 to 99.6%) to an Australian HFMD isolate 1M/AUS/12/2000, surpassing the identity values (98.5 to 98.6%) of isolates from Malaysia (0756-MAA-97) and Singapore (4575/SIN/98). These findings suggest a strain similar to that previously circulating in Australia in 2000 was introduced to the United Kingdom that became an established lineage in its own right. Finally, a European strain isolated from Norway in 2003 (30) shares very close nucleotide identity (99.6%), with subsequent United Kingdom isolates from 2005 to 2006, again suggesting introduction of a new strain into the United Kingdom from other countries. EV71 is constantly changing, with an estimated genomic evolution rate of substitutions per nucleotide (1). Due to the high rate of subclinical infection, a detailed analysis of every wild-type EV71 strain occurring in the United Kingdom is not achievable since the majority of infections are not investigated. The 32 isolates studied here, despite coming from national reference laboratories, may not be completely representative of the national picture. It is therefore not possible to accurately determine the number of introductions of EV71 into the United Kingdom since undiscovered strains could affect phylogenetic relationships. In addition, the role of herd immunity in driving local viral evolution has not been studied. However, despite these limitations, the present study shows the continuous presence of EV71 C1 genogroup in the United Kingdom from 1998 to Our study suggests a global spread of EV71 strains, with the possibility of a repeated introduction of new strains to the United Kingdom, such that three distinct sublineages were present over three time periods, viz., 1998 to 2001, 2001 to 2004, and 2005 to Seven United Kingdom viruses from two time periods (1999 and 2006) were identified as genogroup C2 strains in the present study. The lack of a local sequence intermediate creates a substantial temporal gap between the United Kingdom viruses, which could account for the unique clustering of the five 2006 strains on the dendrogram. However, the phylogenetic analysis suggests that they are more closely descended from the earlier circulating United Kingdom and Australian isolates, while distant genetic relatedness to strains that circulated widely in Australia in 1995, Malaysia and the United States in 1997, and Japan in 1998 is also indicated by the dendrogram. Of interest were two United Kingdom strains (EP/5622/99 and EP/7414/99) showing high sequence identity (96.6 to 99.8%) with five Australian strains (5/MAUS/599, 6F/ AUS/6/99, 7F/AUS/6/99, 2M/AUS/3/99, and 27M/AUS/2/99) from an epidemic in Western Australia (14). In particular, the United Kingdom strains EP/7414/99 and EP/5622/99 were most closely related to an Australian strain (7F/AUS/6/1999) obtained from a patient with meningitis. The United Kingdom strains do not possess the VP1 170 (A3V) substitution that appeared to be associated with increased neurovirulence of EV71 in that study. Further studies are required to ascertain the role of mutations regarding pathogenesis. The present study did not provide any compelling evidence for recombination among United Kingdom isolates; however, it does identify the interesting possibility of a major recombination event in a previously published sequence (22). Although we would note caution, since despite using the currently available GenBank sequences to perform the analysis, there is no independent means to verify these findings. Clearly, the role of recombination in the diversity of enteroviruses, including EV71, deserves more attention and more careful scrutiny of previous published data. To conclude, EV71 C1 genogroup strains are in continuous circulation in the United Kingdom. The pattern of changes in viral lineages suggested global spread with regular introduction of new strains to the United Kingdom that replaced previous endemic strains. Since EV71 has the potential to cause widespread epidemics with fatal complications, its epidemiology and molecular evolution, including the possibility of intertypic and intratypic recombinations, need to be carefully monitored. ACKNOWLEDGMENTS Funding for this study was provided by Guy s and St Thomas Charity grant R We thank Siobhan O Shea for critical appraisal of the manuscript. REFERENCES 1. Brown, B. A., M. S. Oberste, J. P. Alexander, Jr., M. L. Kennett, and M. A. Pallansch Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to J. Virol. 73: Cardosa, M. J., D. Perera, B. A. Brown, D. Cheon, H. M. Chan, K. P. Chan, H. Cho, and P. C. McMinn 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: Chonmaitree, T., M. A. Menegus, E. M. Schervish-Swierkosz, and E. Schwalenstocker Enterovirus 71 infection: report of an outbreak with two cases of paralysis and a review of the literature. Pediatrics 67: Chumakov, M., M. Voroshilova, L. Shindarov, I. Lavrova, L. Gracheva, G. Koroleva, S. Vasilenko, I. Brodvarova, M. Nikolova, S. Gyurova, M. Gacheva, G. Mitov, N. Ninov, E. Tsylka, I. Robinson, M. Frolova, V. Bashkirtsev, L. Martiyanova, and V. Rodin Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch. Virol. 60: Felsenstein, J PHYLIP (phylogeny inference package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle. 6. Herrero, L. J., C. S. Lee, R. J. Hurrelbrink, B. H. Chua, K. B. Chua, and P. C. McMinn Molecular epidemiology of enterovirus 71 in peninsular Malaysia, Arch. Virol. 148: Ho, M., E. R. Chen, K. H. Hsu, S. J. Twu, K. Y. Chen, S. F. Tsai, J. R. Wang, S. R. Shih, et al An epidemic of enterovirus 71 infection in Taiwan. N. Engl. J. Med. 341: Kehle, J., B. Roth, C. Metzger, A. Pfitzner, and G. Enders Molecular characterization of an enterovirus 71 causing neurological disease in Germany. J. Neurovirol. 9: Liu, C. C., H. W. Tseng, S. M. Wang, J. R. Wang, and I. J. Su An outbreak of enterovirus 71 infection in Taiwan, 1998: epidemiologic and clinical manifestations. J. Clin. Virol. 17: 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 Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. 73: Lukashev, V. A Role of recombination in evolution of enteroviruses. Rev. Med. Virol. 15: Lum, L. C., K. T. Wong, S. K. Lam, K. B. Chua, A. Y. Goh, W. L. Lim, B. B. Ong, G. Paul, S. AbuBakar, and M. Lambert Fatal enterovirus 71 encephalomyelitis. J. Pediatr. 133:

9 3200 BIBLE ET AL. J. CLIN. MICROBIOL. 13. McMinn, P., I. Stratov, and L. S. Davis Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin. Infect. Dis. 32: McMinn, P., K. Lindsay, D. Petera, H. M. Chan, K. P. Chan, and M. J. Cardosa Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J. Virol. 75: Mizuta, K., C. Abiko, T. Murata, Y. Matsuzaki, T. Itagaki, K. Sanjoh, M. Sakamoto, S. Hongo, S. Murayama, and K. Hayasaka Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and J. Clin. Microbiol. 43: Page, R. D. M TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12: Pallansch, M. A., and R. P. Roos Enteroviruses: polioviruses, coxsackie-viruses, echoviruses, and newer enteroviruses, p In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed. Lippincott/The Williams & Wilkins Co., Philadelphia, PA. 18. Tu, P. V., N. T. T. Thao, D. Perera, K. H. Truong, N. T. K. Tien, T. C. Thuong, O. M. How, M. J. Cardosa, and P. C. McMinn Epidemiologic and virologic investigation of hand, foot, and mouth disease, Southern Vietnam, Emerg. Infect. Dis. 13: Richter, J., D. Koptides, C. Tryfonos, and C. Christodoulou Molecular typing of enteroviruses associated with viral meningitis in Cyprus, J. Med. Microbiol. 55: Roth, B., M. Enders, A. Arents, A. Pfitzner, and E. Terletskaia-Ladwig Epidemiologic aspects and laboratory features of enterovirus infections in Western Germany, J. Med. Virol. 79: Schmidt, N. J., E. H. Lennette, and H. H. Ho An apparently new enterovirus isolated from patients with disease of the central nervous system. J. Infect. Dis. 129: Shih, S. R., M. S. Ho, K. H. Lin, S. L. Wu, Y. T. Chen, C. N. Wu, T. Y. Lin, L. Y. Chang, K. C. Tsao, H. C. Ning, P. Y. Chang, S. M. Jung, C. Hsueh, and K. S. Chang Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, Virus Res. 68: 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 Enterovirus 71 from fatal and nonfatal cases of hand, foot and mouth disease epidemics in Malaysia, Japan and Taiwan in Jpn. J. Infect. Dis. 52: Shimizu, H., A. Utama, N. Onnimala, C. Li, Z. Li-Bi, M. Yu-Jie, Y. Pongsuwanna, and T. Miyamura Molecular epidemiology of enterovirus 71 infection in the Western Pacific Region. Pediatr. Int. 46: Shindarov, L. M., M. P. Chumakov, M. K. Voroshilova, S. Bojinov, S. M. Vasilenko, I. Iordanov, I. D. Kirov, E. Kamenov, E. V. Leshchinskaya, G. Mitov, I. A. Robinson, S. Sivchev, and S. Staikov Epidemiological, clinical, and pathomorphological characteristics of epidemic poliomyelitislike disease caused by enterovirus 71. J. Hyg. Epidemiol. Microbiol. Immunol. 23: Thompson, J. D., D. G. Higgins, and T. J. Gibson CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: Wang, S. M., C. C. Liu, H. W. Tseng, J. R. Wang, C. C. Huang, Y. J. Chen, Y. J. Yang, S. J. Lin, and T. F. Yeh Clinical spectrum of enterovirus 71 infection in children in southern Taiwan, with an emphasis on neurological complications. Clin. Infect. Dis. 29: Wang, J. R., Y. C. Tuan, H. P. Tsai, J. J. Yan, C. C. Liu, and I. J. Su Change of major genotype of enterovirus 71 in outbreaks of hand-foot-andmouth disease in Taiwan between 1998 and J. Clin. Microbiol. 40: Witsø, E., G. Palacios, K. S. Rønningen, O. Cinek, D. Janowitz, M. Rewers, B. Grinde, and W. I. Lipkin Asymptomatic circulation of HEV71 in Norway. Virus Res. 123: World Health Organization Enterovirus 71 surveillance. Wkly. Epidemiol. Rec. 7: Downloaded from on December 24, 2018 by guest