Emergence, Circulation, and Spatiotemporal Phylogenetic Analysis ofCoxsackievirus A6- and Coxsackievirus A10-Associated Hand, Foot,and Mouth Disease Infections from 2008 to 2012 in Shenzhen, China

Ya-Qing He,a Long Chen,a,b Wen-Bo Xu,c Hong Yang,a Han-Zhong Wang,d Wen-Ping Zong,a Hui-Xia Xian,a Hui-Ling Chen,a

Xiang-Jie Yao,a Zhang-Li Hu,b Min Luo,a Hai-Long Zhang,a Han-Wu Ma,a Jin-Quan Cheng,a Qian-Jin Feng,e De-Jian Zhaoa

Major Infectious Disease Control Key Laboratory, Shenzhen Center for Disease Control and Prevention, Shenzhen, People’s Republic of Chinaa; Shenzhen Key Laboratoryof Marine Bioresource and Eco-environmental Science, College of Life Sciences, Shenzhen University, Shenzhen, People’s Republic of Chinab; WHO WPRO Regional PolioReference Laboratory and State Key Laboratory for Molecular Virology & Genetic Engineering, Institute for Viral Disease Control and Prevention, Chinese Center forDisease Control and Prevention, Beijing, People’s Republic of Chinac; Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of Chinad;Department of Health Statistics and Epidemiology, College of Public Health, Sun Yat-sen University, Guangzhou, People’s Republic of Chinae

Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently beenassociated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics ofnon-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A totalof 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection ofHEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phyloge-netic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes weredetected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommonserotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012.Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteris-tics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating inShenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionarypressure and frequent recombination (3.2 � 10�3 to 6.4 �10�3 substitutions per site per year for HEV71, CV-A6, CV-A16, andCV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the signifi-cance of the epidemiological surveillance of HFMD.

Coxsackievirus A6 (CV-A6) and coxsackievirus A10 (CV-A10)are naked positive single-stranded RNA viruses which belong

to the human enteroviruses (HEVs). HEVs, including poliovirus(PV), coxsackievirus A and B (CV-A and CV-B), echovirus (E),and new human enterovirus (HEV), are among the most commonhuman infectious viruses and mainly infect neonates and youngchildren (1). Based on their molecular characterizations, HEVsinclude the species A to D.

Although most HEV infections are asymptomatic, they cancause a wide range of clinical manifestations ranging from mildsymptoms to fatal disease, such as hand, foot, and mouth disease(HFMD), herpangina, onychomadesis, acute hemorrhagic con-junctivitis, acute respiratory tract infection, aseptic meningitis,encephalitis, myocarditis, and acute flaccid paralysis (1, 2).HFMD infection in children younger than 5 years old typicallypresents as a brief, generally mild, febrile illness with a papulove-sicular rash on the palms and soles and multiple oral ulcers (3).

As a major causative agent of HFMD, epidemic waves of hu-man enterovirus 71 (HEV71) have swept through countries in theAsia-Pacific region since 1997 (4). In mainland China, the HEV71strain was first isolated in 1987 from an HFMD patient withoutneurological symptoms (5). Since a large outbreak of HFMD with405 severe infections and 78 deaths in Taiwan occurred in 1998,HEV71 has become the dominant cause of HFMD, which is prev-alent in mainland China. Three large HEV71 outbreaks resulted in14, 23, and 126 deaths, respectively, in Linyi, Shandong province,

in 2007, Fuyang, Anhui province, in 2008, and Taiwan in 2008(6–8). Severe HFMD with neurological system illness (acute flac-cid paralysis, brainstem encephalitis associated with cardiopul-monary edema) has been mainly caused by HEV71, according to afew large outbreaks of HFMD in the world. In contrast, worldwideepidemiological studies of HFMD showed that CV-A16 and anumber of other HEV-A serotypes usually cause mild self-limitinginfections (9).

In the past few years, HEV-B has been relatively more prevalentthan HEV-A in certain regions. Recently, CV-A6 and CV-A10 ofthe HEV-A serotype have been increasingly associated with infec-tious disease, such as HFMD, herpangina, and onychomadesis(10–16). The prevalences of other HEV-A infections were under-estimated for many years because more attention has been paid to

Received 12 May 2013 Returned for modification 1 July 2013Accepted 12 August 2013

Published ahead of print 21 August 2013

Address correspondence to Ya-Qing He, heyaqing1019@126.com.

Y.-Q.H. and L.C. contributed equally to this study.

Supplemental material may be found for this article at http://dx.doi.org/10.1128/JCM.01231-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.01231-13

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HEV71 and CV-A16 in China. Shenzhen, as a special economiczone in China, is located on the southern coast. Its high popula-tion density, high population mobility, and subtropical environ-ment make Shenzhen an HFMD-prone area. From 2008 to 2012,the detection ratio of non-HEV71 and non-CV-A16 enterovirusesindicated an upward trend by real-time reverse transcription-PCR(RT-PCR) from the sentinel surveillance systems for HFMD(Shenzhen Center for Disease Control and Prevention [CDC]).Therefore, a prospective observational study on the causativeagents of HFMD was performed to clarify the roles of other hu-man enterovirus types, with an emphasis on exploring the preva-lences and genetic characteristics of CV-A6 and CV-A10.

MATERIALS AND METHODSSpecimen collection and study protocol. From 2008 to 2012, sentinelpediatricians were requested to collect clinical specimens from patientspresenting with HFMD. Each specimen along with a standardized reportform was sent to the virology laboratory at the Shenzhen CDC for thedetection of HEVs. The report form recorded information on patientdemographics and clinical findings. According to the diagnostic criteriadefined previously by the Ministry of Health, children were clinically di-agnosed as having HFMD if they had a fever and onset of at least one of thefollowing features: maculopapular or vesicular rash on the palms and/orsoles and vesicles or ulcers in the mouth. Children with serious complica-tions, including encephalitis, meningitis, acute flaccid paralysis, cardiore-spiratory failure, or death, were considered to have severe HFMD. Chil-dren diagnosed with HFMD but without the abovementioned seriouscomplications were classified as having mild HFMD.

A total of 2,411 clinical stool specimens were collected and archived bythe Department of Microbiology at the Shenzhen CDC between 2008 and2012 (590 in 2008, 299 in 2009, 458 in 2010, 628 in 2011, and 436 in 2012).HEV RNA was detected by real-time RT-PCR and reverse transcriptionseminested PCR (RT-snPCR) (17, 18). The study was performed accord-ing to the Declaration of Helsinki II and was approved by the ethics com-mittees of Shenzhen Children’s Hospital, Long Gang Center Hospital, andPing Shan People’s Hospital. Written informed consent was obtainedfrom all patients or their caretakers.

Viral RNA extraction and real-time RT-PCR. Specimen processingwas performed as previously described (18). Viral RNA was extractedfrom 200 �l supernatant using the High Pure viral RNA kit (Roche, Ger-many) and was subsequently used for HEV71 and CV-A16 detection us-ing a real-time RT-PCR kit (Shenzhen Taitai Genomics, Inc., China). Aknown-negative stool specimen was included in each extraction run, andthe extract was tested by real-time RT-PCR along with the clinical speci-mens to monitor for cross-contamination. The runs that occasionallyyielded false-positive results occasionally were excluded from the analysis.

RT-PCR, RT-snPCR amplification, and sequencing. Samples thatwere positive for HEV71 or CV-A16 by real-time RT-PCR were randomlyselected to amplify the entire VP1 gene for phylogenetic analysis usingRT-PCR with specific primers (6). For specimens that were negative forboth HEV71 and CV-A16, the typing of HEVs based on the VP1 gene wasperformed by three rounds of independent reverse transcription-semin-ested PCR (RT-snPCR). First, cDNA was synthesized in a 10-�l volumereaction mixture using the PrimeScript II 1st Strand cDNA synthesis kit(TaKaRa, Japan).

Next, a 3-�l cDNA solution was used as a template in each round ofRT-snPCR. The first round of RT-snPCR based on the complete VP1 genewas performed to detect HEV-A using the primers HEVAS1495,HEVAR2C, and HEVAR2807 (19). The second round of RT-snPCR basedon the complete VP1 gene was performed to detect HEV-B using theprimers HEVBS1695, EV2C, and HEVBR132 (20). The third round ofRT-snPCR based on the partial VP1 gene was performed to detect otherHEVs using two pairs of CODEHOP primers, 224/222 and AN89/AN88(18). Amplified DNA was purified using a commercial kit (catalog no.

D823A, MiniBEST agarose gel DNA extraction kit version 3.0; TaKaRa,Japan) and sequenced by the ABI PrismTM 3730xl DNA analyzer usingthe BigDye Terminator version 3.1 cycle sequencing kit (Applied Biosys-tems). A contig was assembled with forward and reverse nucleotide se-quences.

Identification of HEVs serotypes and phylogenetic analyses. Assem-bled sequences were used to identify the serotypes of HEV strains usingthe online Enterovirus Genotyping Tool (http://www.rivm.nl/mpf/enterovirus/typingtool) or a BLAST search. The nucleotide sequences ofHEV strains assigned to serotypes HEV71, CV-A6, CV-A16, and CV-A10were compared with homologous sequences available in GenBank toidentify variants and analyze the phylogenetic relationships with strainsthat are circulating globally. Multiple sequence alignments were per-formed by ClustalX 2.0.12, which is available in the European Bioinfor-matics Institute. All reference sequences were derived from GenBank. Theneighbor-joining (NJ), maximum-likelihood (ML), and BayesianMarkov chain Monte Carlo (BMCMC) methods were used to compara-tively analyze the phylogenesis of the partial VP1 genes of CV-A6 andCV-A10 using MEGA 5.05 (21) and BEAST 1.7.40 (22). The spatiotem-poral evolution of these four pathogens was inferred by BMCMC analysis.Bayesian analyses were performed using a relaxed molecular clock model(the uncorrelated lognormal distributed model [UCLD]) (23). The anal-yses were independently performed using the Hasegawa-Kishino-Yano(HKY) (24) and general time-reversible (GTR) (25) nucleotide substitu-tion models, with a gamma-distributed among-site rate variation withfour rate categories. BMCMC analyses were repeated using the constantsize and exponential growth models in order to investigate the degree towhich the dating estimates were affected by the demographic modelchosen.

Statistical analyses. Data were analyzed using the statistical softwareSPSS version 18.0. The statistical differences of the male/female and se-vere/mild ratios between different HEVs serotypes were tested by chi-square test. An analysis of variance was used to compare the means of ages.A P value of �0.05 was regarded as statistically significant.

Nucleotide sequence accession numbers. The sequences determinedin this study (n � 779) were submitted to GenBank/EMBL/DDBJ underthe accession numbers KC866623 to KC867102, JX154899 to JX155009,and JX473292 to JX473479.

RESULTSThe distribution of HEV infection. Among the 2,411 totalHFMD cases, 1,803 (74.8%) were positive for HEVs. A total of 28different serotypes were identified by sequencing between 2008and 2012 (Table 1; see also Table S1 in the supplemental material),in which 9 serotypes belonged to HEV-A species, 18 serotypesbelonged to HEV-B species, one serotype belonged to HEV-C spe-cies, and no HEV-D species were detected. HEV-A species werethe most prevalent (96.7% [1,744/1,803]), followed by HEV-Bspecies (3.2% [57/1,803]).

During 2008 and 2009, the most prevalent serotype wasHEV71, followed by CV-A16. Although HEV71 remained themost common serotype between 2010 and 2012, CV-A6 replacedCV-A16 as the second most common HEV. Within 5 years, onlyCV-A6 showed a trend of increasing prevalence (from 0.5% to27.4% between 2008 and 2012), while CV-A10 strains circulatedat a relatively low prevalence (�7%). CV-A6 has recently emergedas a serotype circulating with increasing frequency in Shenzhen,and in 2012, it turned into as common a causative agent of HFMDas HEV71 and CV-A16.

Epidemiological data. The HEV infections mainly occurredbetween April and September during the years 2008 and 2012 (Fig.1A to E). HEV71 infections generally occurred throughout theyear between 2008 and 2012 but occurred mainly during the warm

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seasons. The proportion of HEV71 infections (among all HEVs)significantly dropped in 2012 relative to the previous 4 years. CV-A16 infections occurred throughout the year during 2008 and2009, but the proportion of CV-A16 infections obviously declined

in 2010 and then increased gradually from 2011 to 2012. Contraryto HEV71, CV-A6 infections mainly occurred during the autumnand winter.

Statistical analysis indicated that the mean age of CV-A6- or

TABLE 1 Distributions of HEV infections from HFMD patients between 2008 and 2012

YrNo. (%) ofpositive cases

Prevalence of serotypes by ranka:

1 2 3 4 5

SerotypeNo. (%)of cases Serotype

No. (%)of cases Serotype

No. (%)of cases Serotype

No. (%)of cases Serotype

No. (%)of cases

2008 407 (69.0) HEV71 240 (59.0) CV-A16 147 (36.1) CV-B3 5 (1.2) CV-A10 3 (0.7) CV-A4 3 (0.7)2009 244 (81.6) HEV71 131 (53.7) CV-A16 71 (29.1) CV-A6 20 (8.2) CV-A10 15 (6.1) CV-A5 2 (0.8)2010 330 (72.1) HEV71 233 (70.6) CV-A6 37 (11.2) CV-A16 20 (6.1) CV-A10 13 (3.9) CV-A4 4 (1.2)2011 472 (75.2) HEV71 302 (64.0) CV-A6 79 (16.7) CV-A16 57 (12.1) CV-A10 19 (4.0) CV-B2 3 (0.6)2012 350 (80.3) HEV71 121 (34.6) CV-A6 96 (27.4) CV-A16 87 (24.9) CV-A2 12 (3.4) CV-A10 10 (2.9)a The other ranks are listed in Table S1 in the supplemental material.

FIG 1 Distribution of the number of human enterovirus-positive specimens and prevalent serotypes by month between 2008 and 2012 in Shenzhen, China. (A)2008; (B) 2009; (C) 2010; (D) 2011; (E) 2012. CV-A, coxsackievirus A; HEV, human enterovirus.

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CV-A10-infected patients was lower than the mean age of thoseinfected with HEV71 in 2009, 2010, and 2012 (P � 0.05) (Table 2).The male/female ratio of CV-A16-infected patients was higherthan that of patients infected with other HEVs in 2010 (P �0.025), and the male/female ratio of CV-A6-infected patients washigher than that of patients infected with other HEVs in 2012 (P �0.001). The majority of HEV infection cases (78.5% [1,416/1,803]) presented with mild symptoms. However, the severe/mildratio was higher in HEV71-infected patients than in those whotested positive for CV-A6, CV-A16, or CV-A10 (P � 0.001) dur-ing 2009 and 2012. Severe symptoms included accelerated breath-ing, severe vomiting, muscle twitches, abnormal eye movements,brainstem encephalitis, and aseptic meningitis. In patients withsevere symptoms with HEV71, CV-A6, CV-A16, and CV-A10 in-fections, the common symptoms included accelerated breathingand muscle twitches (data not shown).

Spatiotemporal phylogenetic analysis. Phylogenetic analysisof complete VP1 sequences showed that all HEV71 strains (n �192) identified in this study belonged to subgenogroup C4. A highdegree of similarity (93.4% to 100%) was observed between thesequences. Since 1998, the subgenogroup C4 has been responsiblefor almost all HEV71 infections in mainland China. Maximum-likelihood phylogenetic analysis of the Chinese HEV71 sequencesindicated that there were 2 stages of HEV71 circulation in main-land China between 1998 and 2012 (see Fig. S1 in the supplemen-tal material). The HEV71 strains isolated from Shenzhen showeda shift from evolutionary branch C4b to C4a between 2003 and2004 (see Fig. S1 in the supplemental material). We estimated thatthe common ancestor HEV71 likely emerged at the beginning ofthe 20th century, and its evolutionary rate was estimated to be 3.2�10�3 to 3.3 �10�3 substitutions per site per year (see Table S2 in

the supplemental material). Comparing the estimated time of or-igin with the dates of first detection of each subgenotype, we foundthat divergent B and C subgenotypes circulated recessively forapproximately 1 to 7 years before causing large HFMD outbreaks(see Table S2 in the supplemental material). Phylogenetic analysisbased on complete VP1 sequences indicated that CV-A16 strains(n � 102) in this study were grouped with B1a and B1b with 86.9to 100% identity (see Fig. S2 in the supplemental material). Theprevalent subgenotypes B1a and B1b mainly cocirculated in theAsia-Pacific regions. CV-A16 strains isolated from Shenzhen re-vealed the cocirculation of B1a and B1b from 2005 to 2012. Thecommon ancestor CV-A16 likely emerged between 1804 and 1806(exponential growth model), and its evolutionary rate was esti-mated to be 4.0 �10�3 to 4.1 �10�3 substitutions per site per year(see Table S3 in the supplemental material). Genotype B2 anddivergent B1 subgenotypes were estimated to circulate recessivelyfor 3 to 4 years before causing large HFMD outbreaks.

The NJ, ML, and BMCMC phylogenetic trees of CV-A6 basedon partial VP1 sequences (269 nt) showed the same topology (Fig.2; see also Fig. S3 and S4 in the supplemental material). Genotyp-ing for CV-A6 strains was performed in a similar fashion as thatwhich was described for HEV71 (26). A difference of �15% in theVP1 genes was used to distinguish between the different geno-types. Accordingly, the CV-A6 strains were classified into six ma-jor clusters, denoted A, B, C, D, E, and F (Fig. 2). The completeVP1 genes of 142 CV-A6 strains in this study showed 73.2% to99.9% similarity. All CV-A6 strains (n � 234) determined in thisstudy between 2008 and 2012 were categorized into three geno-groups, the majority of which clustered with genogroup D, 8strains (isolated from 2008 to 2012) clustered with genogroup C,and only one variant strain, JB143090087 (isolated in 2009),grouped with genogroup B. A majority of the CV-A6 strains iso-lated from Shenzhen between 2008 and 2012 and from Japan in2011 displayed a close genetic relationship with 2008 Finnishstrains associated with an HFMD outbreak, 2008 Spanish strainsassociated with onychomadesis after HFMD, and 2010 Frenchstrains associated with an HFMD/herpangina outbreak (10, 12,15, 16). The genetic characteristics of the CV-A6 strains isolated inChina displayed geographical differences: the predominant geno-type of CV-A6 was genogroup D between 2008 and 2012 in Shen-zhen, whereas the prevalent genotype was B in other regions inChina. Therefore, we inferred that the Chinese CV-A6 strains haddifferent geographical origins, and CV-A6 strains from Shenzhenlikely originated in Europe. The most common ancestor of CV-A6emerged between 1857 and 1860 (exponential growth model),and its evolutionary rate was estimated to be 4.5 �10�3 to4.6 �10�3 substitutions per site per year (Table 3). The prevalentgenotypes C and D were estimated to circulate recessively for 5 to6 years before causing infectious diseases.

The NJ, ML, and BMCMC phylogenetic trees of CV-A10 basedon partial VP1 sequences (264 nt) also displayed the same topol-ogy (Fig. 2; see also Fig. S5 and S6 in the supplemental material).CV-A10 strains were classified into seven major clusters based onthe criteria described above, designated A, B, C, D, E, F, and G(Fig. 2). The complete VP1 sequences of 55 CV-A10 strains in thisstudy showed 81.3% to 100% similarity. All CV-A10 strains (n �60) determined in this study between 2008 and 2012 were in-cluded in genogroup C except for two variant strains,JB143090148 and JB143090155 (isolated in 2009), which wereclustered in genogroup F. The majority of CV-A10 strains isolated

TABLE 2 Demographics associated with the different HEVs from 2008to 2012

Demographic andyears of infection

Results by HEV serotype

PEV71 CV-A6 CV-A16 CV-A10

Mean age � SD (yr)2008 3.0 � 1.6 1.0 � 0 2.9 � 1.4 1.6 � 0.9 0.0892009 2.0 � 1.3 1.6 � 1.1 2.2 � 1.5 1.4 � 0.5 0.0242010 2.1 � 1.4 1.8 � 0.9 2.1 � 1.5 1.1 � 0.5 0.0142011 2.2 � 1.3 2.1 � 1.5 2.1 � 1.8 1.9 � 1.4 0.5892012 2.3 � 1.4 1.8 � 1.0 2.3 � 1.5 1.8 � 0.9 0.014

Male/female ratio2008 2.24 NAa 1.83 2.00 0.4892009 2.05 2.33 3.18 2.00 0.6022010 1.33 2.08 5.67 0.63 0.0252011 1.54 1.39 1.48 2.80 0.6662012 1.88 2.69 1.72 1.5 �0.001

Severe/mild ratiob

2008 0.01 0 0 0 0.3982009 0.72 0.33 0.16 0.07 �0.0012010 0.73 0.06 0.11 0 �0.0012011 0.75 0.10 0.14 0.19 �0.0012012 0.51 0.08 0.09 0 �0.001

a NA, not available.b Patients presenting with hand, foot, and mouth disease with serious complications,including encephalitis, meningitis, acute flaccid paralysis, cardiorespiratory failure, ordeath, were considered to have severe cases. Patients without the abovementionedserious complications were classified as having mild cases.

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from Shenzhen between 2008 and 2012 had a close genetic rela-tionship with the 2007 Slovakian strains and the 2008 Spanishstrains associated with onychomadesis after HFMD (15); how-ever, they displayed a distant genetic relationship with the 2008Finnish strains associated with an HFMD outbreak and the 2010French strains associated with an HFMD/herpangina outbreak(10, 12). CV-A10 strains circulated in China with a low preva-lence, and the genetic characteristics of the CV-A10 strains iso-lated from Shenzhen were different from those isolated from otherregions in China. The most common ancestor of CV-A10emerged almost at the same time as HEV71, and its evolutionaryrate was estimated to be 6.2 �10�3 to 6.4 �10�3 substitutions persite per year (Table 3). Genotypes B, C, D, E, and F were estimatedto circulate recessively for 2 to 11 years before causing infectiousdiseases.

DISCUSSION

As a common infectious disease, HFMD is a serious threat topublic health. Numerous large epidemics of HFMD have occurredmainly in eastern and southeastern Asian countries and regions inthe past decades, with HEV71 being the most commonly respon-sible causative agent. In recent years, however, some uncommonHEVs emerged as being occasionally prevalent. CV-A6 and CV-

A10 especially began to cocirculate with increasingly frequency insome European countries, which resulted in these serotypes be-coming as common causes of HFMD as were HEV71 and CV-A16in certain regions (10–16). In mainland China, the surveillance ofHFMD has been focused mainly on HEV71 and CV-A16. There-fore, little is known about the pathogenic roles of other HEVs,their geographic distributions, and epidemiological data. Ourprospective study is the first to provide a 5-year surveillance ofHFMD over the past 5 years in China. The results demonstratedthat CV-A6 and CV-A10 emerged and cocirculated with a varietyof other HEVs in Shenzhen, although HEV71 remained the majorpathogen. As an emerging pathogen, CV-A6 increasingly becameas common a causative agent of HFMD in Shenzhen as wasHEV71. Although HEV71 and CV-A6 have been the main patho-gens of HFMD in Shenzhen in recent years, HEV71 and CV-A16remained the major causes of HFMD in other regions in mainlandChina.

HEV-A species were the most common HEV types (94.3% to98.8% between 2008 and 2012) among the pathogens causingHFMD in Shenzhen, followed by HEV-B and HEV-C species,while no HEV-D species were detected. However, HEV-B specieswere the most frequent HEV types in healthy children (�5 years)and in an aquatic environment between 2010 and 2011 in Shen-

FIG 2 The ML phylogenetic trees based on partial VP1 sequences of global CV-A6 (269 nt) (A) and CV-A10 isolates (264 nt) (B). The nucleotide substitutionmodel used was the general time-reversible method. The sequences of this study are labeled with Œ (indicating a single sequence) or Š (indicating multiplesequences). The trees were midpoint rooted, and bootstrap support values of �70% (1,000 bootstrap replicates) were not indicated. The reference sequences arelabeled as GenBank accession no.-location-year.

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zhen (27, 35). These results suggest that the prevalences of HEVsin Shenzhen have been different in different environments.

Previous studies demonstrated that HEV71 was more likely tocause serious complications than other HEVs and often led toacute flaccid paralysis, brainstem encephalitis associated with car-diopulmonary edema, and death. However, in our study, in pa-tients with severe HEV71 or CV-A16 infections, the shared com-mon symptoms were accelerated breathing and muscle twitches.Phylogenetic analysis showed that little variation appeared in theChinese HEV71 and CV-A16 strains; Chinese HEV71 strains havebeen the C4 genotype since 1998, and Chinese CV-A16 strainshave had subgenotypes B1a and B1b since 1999. Further studiesshould be intensified to clarify the relationship between patho-genic features and the genetic characteristics of the pathogens.

In consideration of further evolutionary dynamics analysis ofthe pathogens, three RT-snPCR methods were chosen to identifythe causative agents of HFMD in this study, which not only en-sured the high sensitivity of detection but also amplified completeVP1 genes. Spatiotemporal phylogenetic analysis of CV-A6 andCV-A10 strains revealed genetic diversity. There was great geneticvariation among the CV-A6 and CV-A10 strains from differentregions in mainland China. We inferred that CV-A6 strains circu-lating in Shenzhen likely originated in Europe. The prevalent ge-notypes C and D were estimated to circulate for 5 to 6 years beforecausing infectious diseases. Therefore, we must be on the alert forthe possibility that CV-A6 will cause an HFMD outbreak in thenext few years in China. Although the genotyping of CV-A6 andCV-A10 was performed by three statistical methods, a robust spa-tiotemporal phylogenetic analysis of CV-A6 and CV-A10 basedon complete VP1 sequences is needed.

The molecular evolutionary rates of several human enterovirusserotypes have been estimated to be 3 � 10�3 to 9 � 10�3 substi-tutions/site/year (28–31). Since RNA viruses have an estimatedmutation rate of between 10�3 and 10�5 substitutions/site/gener-ation (32), this suggests that the molecular evolutionary rates ofHEVs are higher than those of the other RNA viruses. HEVs un-

derwent rapid variation under evolutionary pressure and frequentrecombination (8, 28, 33, 34). For this reason, the pathogens ofHFMD have evolved into multiple genotypes that have served as abottleneck for vaccine development against HEVs. In summary,our study indicated that a variety of HEV serotypes have beeninvolved in HFMD infections, according to our 5-year surveil-lance in China. Identifying the causative agents of HFMD has beenchallenging. Continuing surveillance is needed to identify otherunusual strains.

ACKNOWLEDGMENTS

This work was supported in part by grants from the National NaturalScience Foundation of China (no. 31170874).

We are grateful to the pediatricians who are from the sentinel surveil-lance system for HFMD in Shenzhen, China.

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TABLE 3 Evolutionary characteristics of CV-A6, CV-A10, and their genogroups

tMRCA by serotype and othercharacteristicsa

Data by BMCMC growth model (mean [95% CI])b:

Constant size Exponential growth

HKY � �4 GTR � �4 HKY � �4 GTR � �4

tMRCA CV-A6 (1949) 1867 (1780–1949) 1866 (1780–1949) 1857 (1780–1949) 1860 (1760–1949)Evolutionary ratec 4.5 (2.8–6.5) 4.5 (2.8–6.7) 4.5 (2.8–6.9) 4.4 (2.7–6.5)Coefficient of variation 0.41 (0–0.74) 0.40 (0–0.76) 0.42 (0–0.78) 0.39 (0–0.73)

tMRCA B (1992) 1981 (1972–1988) 1981 (1971–1988) 1981 (1972–1989) 1980 (1971–1988)tMRCA C (1999) 1994 (1990–1997) 1994 (1990–1997) 1994 (1990–1997) 1994 (1990–1997)tMRCA D (2008) 2002 (1998–2005) 2003 (1998–2005) 2003 (1998–2005) 2003 (1998–2005)tMRCA CV-A10 (1950) 1907 (1866–1949) 1906 (1861–1946) 1905 (1858–1945) 1903 (1857–1944)

Evolutionary ratec 6.2 (4.0–8.6) 6.4 (4.0–8.9) 6.3 (4.2–8.8) 6.2 (4.2–8.6)Coefficient of variation 0.37 (0.02–0.69) 0.39 (0–0.68) 0.36 (0–0.67) 0.35 (0–0.66)

tMRCA B (1999) 1994 (1990–1998) 1994 (1990–1998) 1994 (1990–1998) 1994 (1990–1998)tMRCA C (2001) 1990 (1983–1996) 1990 (1984–1996) 1990 (1983–1996) 1990 (1984–1996)tMRCA D (2003) 1997 (1991–2001) 1997 (1991–2002) 1997 (1991–2002) 1996 (1990–2002)tMRCA E (2003) 2001 (1999–2003) 2001 (1999–2003) 2001 (2000–2003) 2001 (1999–2003)tMRCA F (2004) 1999 (1995–2003) 1999 (1995–2003) 1999 (1995–2003) 1999 (1995–2003)a The year in parentheses is the the earliest time of identification of the serotype or genogroup. tMRCA, time to the most common ancestor.b Values are estimated years of emergence of the most common ancestor unless otherwise indicated. BMCMC, Bayesian Markov chain Monte Carlo method; HKY, Hasegawa-Kishino-Yano method; GTR, general time-reversible method; CI, confidence interval; �4, a gamma-distributed among-sites rate variation with four rate categories that allows ratevariation between sites in the associated alignment.c Evolutionary rates are expressed as �10�3 substitutions per site per year.

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