Characterization of Full-Length Enterovirus 71 Strains
from Severe and Mild Disease Patients in Northeastern
Xiaomei Wang1,2., Chunfeng Zhu1., Wanguo Bao3, Ke Zhao1, Junqi Niu2*, Xiao-Fang Yu1*,
1Institute of Virology and AIDS Research, First Hospital of Jilin University, Changchun, Jilin Province, China, 2Department of Hepatology, First Hospital of Jilin University,
Changchun, Jilin Province, China, 3Department of Infectious Diseases, First Hospital of Jilin University, Changchun, Jilin Province, China
Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood
infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China,
including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern
region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD
patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from
Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified
as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5,
EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71
C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in
different regions. The 59UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree
of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients
in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that
all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence
and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in
Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications
for vaccine development that would address the increasing prevalence of HFMD.
Citation: Wang X, Zhu C, Bao W, Zhao K, Niu J, et al. (2012) Characterization of Full-Length Enterovirus 71 Strains from Severe and Mild Disease Patients in
Northeastern China. PLoS ONE 7(3): e32405. doi:10.1371/journal.pone.0032405
Editor: Krzysztof Pyrc, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Poland
Received November 20, 2011; Accepted January 30, 2012; Published March 29, 2012
Copyright: ? 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by funding from the Specialized Research Fund for the Doctoral Program of Higher Education and Jilin University
(No200903258) to Wenyan Zhang, and a grant from the Key Laboratory of Molecular Virology, Jilin Province (20102209), China. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (WZ); email@example.com (X-FY); firstname.lastname@example.org(JN)
. These authors contributed equally to this work.
Hand, foot and mouth disease (HFMD) is a common, mild and
self-limiting rash-associated illness in children, with coxsackievirus
A16 (CA16) or enterovirus 71 (EV71) as the causative agent [1,2].
Since first being described in California in 1969 , EV71 has
been reported to be responsible for many large outbreaks all over
the world, including outbreaks that occurred in Malaysia in 1997
, Taiwan in 1998 , Singapore in 2000 , Japan in 1997 and
2000 , and Shandong and Fuyang of China in 2007  and
2008 , respectively. Generally, the outbreaks were associated
with severe neurologic disease, such as acute flaccid paralysis,
pulmonary edema, myocarditis, and fatal encephalitis.
EV71 is a member of the human enterovirus group A (HEV-A)
and contains a positive, single-stranded RNA genome of
approximately 7500 bases and a single open reading frame
followed by a poly A tract [9,10]. The viral genome contains 59-
and 39-untranslated regions (UTRs) that are essential for viral
expression and replication. The genome encodes for a single, large
polyprotein that is composed of four capsid proteins, VP1 to VP4,
and seven nonstructural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D
. The EV71 viruses were classified into three independent
lineages, A, B, and C, based on the structural VP1 gene; each
group has at least 15% divergence from the others . Group A
consists of one member, the prototype BrCr strain. The B group,
which has been predominant in Malaysia and Singapore, was
separated into subgroups B1 to B5. The C group, which has been
predominant in east Asia, contained subgroups C1 to C5 .
EV71 has a high mutation rate due to low-fidelity replication and
frequent recombination .
Since the 1980s, large and small EV71 epidemics caused by
distinct genotypes have occurred in Asian countries and in regions
sharing trade with China . EV71 epidemics have been
reported in Jilin Province since 2006, and the incidence of HFMD
has increased annually. Here, we report the clinical and molecular
characteristics of EV71-infected patients from Changchun, China
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in 2010. The complete genomes of seven EV71 strains from mild
and severe patients were sequenced and analyzed along with
summarized clinical information. The results of this study
demonstrated that a combination of intratypic and intertypic
recombination involving multiple HEV-A strains occurred within
all seven Changchun EV71 sequences. This suggests that
recombination may be one of the potential reasons for persistent
infection and emerging outbreaks in China. Moreover, all seven
Changchun strains always formed a single cluster with a high
bootstrap value in various phylogenetic analyses of different
genomic regions, suggesting that all seven strains originated from
one single common ancestor, regardless of whether they occurred
in fatal or non-fatal cases; thus, we could not see any correlation
between genome and virulence.
The clinical characteristics of seven patients
Seven throat swabs were chosen from 84 patients who were
identified as EV71-positive by a diagnostic kit (DAAN Gene Co.,
Ltd. of Sun Yat-Sen University). These seven patients from whom
the EV71 was identified were young children (median age of 23
months; ranged from 7 months to 5 years): three were males, and
four were females. All clinical information was obtained from the
medical records of the First Hospital of Jilin University and is
summarized in Table 1. All patients presented with HFMD,
manifested by fever, oral ulcers and vesicular on the hand, and in
some cases, buttocks and knees. Four cases, including those
associated with mild central nervous system (CNS) symptoms such
as vomiting, myoclonic jerk, and irritability, were cured after an
average of 6 days of hospitalization. One case (Changchun063)
was complicated by irregular respiration, tachypnea (50 times/
min) and encephalitis, which resulted in placing the patient on a
ventilator; the patient subsequently recovered after 8 days of
treatment. Two cases (Changchun014 and103) were complicated
by tachypnea, tachycardia, hypotension, pulmonary edema, severe
oliguria and encephalitis; these complications proved fatal in the
patients after 2 days of hospitalization.
Using the poliovirus 1 sequence as an outlier, a phylogenetic
analysis of the complete genome sequences for the HEV-A strains
with these seven samples revealed that the seven Changchun
viruses were clustered into the EV71 C4 subtype; this subtype was
more closely related to subtype B but not to subtype C (Fig. 1A),
although the bootstrap value was 60, lower than 70, which could
not be considered significant. The phylogenetic tree based on the
VP1 gene sequence showed that the seven Changchun strains
were clustered into the subtype C4 lineage; which closely related to
subtype C in this region (Fig. 1B). The incongruent phylogenetic
relationships observed between the complete genome and VP1
sequences suggest that possible recombination events had occurred
in the seven EV71 and SHZH98 strains.
Seven EV71 isolated from HFMD patients in Changchun
are circulating, recombinant strains
To further characterize the genomic features of the seven strains
isolated from Changchun in 2010, each Changchun representative
strain of EV71 was analyzed by similarity plot and bootscan
analysis against other enteroviruses from HEV-A as reference
DQ341359-SAR-98 were selected to represent EV71 type A, B
and C, respectively, while poliovirus 1 was used as an outlier.
EV68 was also included as another outlier to enhance the results.
As a result, all Changchun EV71 genomes displayed intertypic and
intratypic recombination involving multiple type A HEV (See
Fig. 2, showing the Changchun011 strain from a mild case without
CNS symptom, the Changchun077 strain from the CNS
symptoms case and the Changchun103 strain from a fatal case).
The recombination patterns of the other four strains are similar to
those of Changchun011 (Fig. 2A) and 103 (Fig. 2C). Only
Changchun077 had a specific recombinant involving CA4 among
P2 region. In the similarity plot analysis, the sequences of the seven
EV71 strains showed high similarity (77%–100%) to the EV71
genotype C strain (DQ341359-SAR-98) based on the P1 region.
Low similarity (71% and 86%) to the EV71 genotype B and CA5
strains was alternatively noted in the Changchun EV71 strains
among the P2 and partial P3 regions. Before position 500 and after
position 5860, locations that correspond to the 59UTR and 3D
regions, respectively, the seven EV71 strains showed no significant
similarity to any reference sequence. In the bootscan analysis, the
results again indicated that P1 regions of all seven Changchun
sequences were originated from EV71 type C. In the following
analysis of the P2 and partial P3 regions, however, high bootstrap
values showed that the seven EV71 strains contained alternative
regions that were closely related to the EV71 genotype B and CA5
strains. For the 3D region, bootscan analysis did not support any
clustering of the seven EV71 strains. These findings indicated that
intertype and intratype recombination events occurred in the P2
and P3 regions.
Further analysis of the 59UTR and 3D regions
The results described previously showed that no obvious
parental sequence was found for 59UTR and 3D regions of the
seven Changchun EV71 strains, therefore, we carefully examined
the 59UTR and 3D regions by bootscanning and phylogenetic tree
analyses. A more detailed bootscanning of the 59UTR of the seven
strains with a smaller window of 100 bp was performed, and this
analysis showed a low bootstrap with any reference sequence
(Fig. 3A). Phylogenetic analysis of the 744-bp fragment of the
59UTR region is consistent with the bootscanning results, showing
that the sequence did not form a cluster with any specific virus
(Fig. 3B). A more detailed bootscanning of the 3D region within
the seven strains using a smaller window of 200 bp was performed;
it showed a dominant CA16 sequence in the middle (Fig. 3C).
Phylogenetic analysis of the 1386-bp fragment of the 3D region
from the seven EV71 strains is consistent with the bootscanning
results, showing a single cluster that was closely related to CA16
Further analysis of the P1, P2, 2A and 3C regions
We further analyzed the phylogenetic trees of P1 (VP4-VP1), P2
(2A–2C) and other regions of the seven Changchun strains.
Phylogenetic analysis showed that the seven Changchun strains
were clustered with the EV71 genotype C strains for P1 region
(Fig. 4A), but with EV71 genotype B strains for P2 region (Fig. 4B).
The 2A region of the Changchun strains was related to the
sequence of the EV71 genotype C strains (Fig. 4C). The 2C and
3A regions of the strains were closely related to CA5 and EV71B
respectively (data not shown), although the bootstrap value was
very low. Moreover, the 3C region of the Changchun strains was
related to the sequence of CA5 (Fig. 4D). These results further
confirmed that the Changchun strains are complex recombinants
involving multiple HEV-A strains.
Phylogenetic analysis of the seven Changchun strains
with other strains in China
To assess and compare the molecular characteristics of the
seven Changchun isolates with the EV71 strains from the other
Characterization of Enterovirus 71
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Table 1. Clinical manifestations of 7 enterovirus 71 infected patients.
No. of Sample
Sex Male FemaleMaleMale Female FemaleFemale
Age 5Y1Y 7M3Y 2Y 11M9M
Clinical manifestationFeverFever FeverFever FeverFeverFever
Skin vesicular TachycardiaSkin vesicularSkin vesicular Skin vesicular Skin vesicular Skin vesicular
Oral ulcerHypotensionOral ulcer Oral ulcerOral ulcerOral ulcer Oral ulcer
Lethargy Irregular respiration VomitingIrritability Myoclonic jerk
Oliguria Tachypnea Myoclonic jerkLethargy
ComplicationNo Encephalitis EncephalitisNoNo Encephalitis No
Diagnosis HFMDHFMDHFMD HFMDHFMD HFMDHFMD
Days of fever2243332
Days of hospitalization6285824
Underlying diseaseNoNo NoNo NoNo No
OutcomeRecoveryDeath RecoveryRecovery RecoveryDeath Recovery
Figure 1. Phylogenetic analysis of the complete genome and VP1 protein-coding region of the Changchun strains. Phylogenetic trees
were generated by the neighbor-joining method with 1000 bootstraps for 7 representative Changchun strains and other EV71 strains of known
subgenotypes. The poliovirus 1 strain was used as the outlier. The & icon indicates the fatal cases; m indicates the severe cases;Nindicates the mild
cases. A: Phylogenetic tree based on the whole genome sequences. B: Phylogenetic tree based on the VP1 region (891 bp).
Characterization of Enterovirus 71
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Characterization of Enterovirus 71
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provinces in mainland China, phylogenetic trees were constructed
using the complete genome as well as partial regions (Fig. 5). The
outlier group was not used here because all of the China strains
used here were clustered as subtype C4. GenBank database
sequences of 34 epidemic strains from Shenzhen, Henan, Fuyang
and other regions of China were included for comparison. When
comparing the complete genome (Fig. 5A), structural protein VP1
genes (Fig. 5B), 59-UTR (Fig. 5C) nonstructural protein P2 genes
(Fig. 5D) and region phylogenetic trees, the likelihood of
Changchun strains clustering with the LN009 (Liaoning),
NBChina01 (Ningbo) and Henan2 strains revealed the possible
evolution of Changchun strains from these three reference strains.
However, according to the phylogenetic analysis, strains from both
severe and mild HFMD patients kept mixing together, thus it was
hard to determine the relationship between viral sequence and
virulence. Interestingly, the 3C regions of the majority of EV71
strains formed a large cluster (data not shown). We inferred that, as
a protease, 3C protein has to precisely recognize cutting sites
within the polyprotein; thus, 3C has to be conservative to maintain
Molecular characterizations of circulating strains in central and
southern China have been well studied [8,14,15]. However, little
has been reported on the genetic characteristics of EV71 strains
circulating in the northeastern region of China. In this study, we
examined for the first time seven EV71 strains isolated from
severe- or mild-diseased patients in 2010 in Changchun by
phylogenetic tree, bootscan and similarity plot analyses. C4 was
previously identified as the most prominent EV71 subgenotype
circulating in China . Phylogenetic analyses on the complete
genome and VP1 sequences showed that all Changchun strains
belong to the C4 subgenotype (Fig. 1). This result suggests that
Changchun EV71 did not evolve independently. Complete
Figure 3. Bootscan and phylogenetic analyses of partial genomes from the Changchun strains. A: Identification of the recombinant
sequences in the 59-UTR region of the Changchun011 genome. The window size of 100 nucleotides slides in increments of 20 nucleotides at a time. B:
The neighbor-joining tree was established from alignments of the entire 59-UTR region for the seven Changchun strains. C: Bootscan analysis of a 3D
region in the Changchun011 genome. The window size included 200 nucleotides slides in increments of 20 nucleotides at a time. D: The neighbor-
joining tree was established from sequence alignments of the entire 3D region for the seven Changchun strains. The & icon indicates the fatal cases;
m indicates the severe cases;Nindicates the mild cases.
Figure 2. Identification of recombination in the Changchun strains. A: Similarity plot and bootscan analysis for Changchun011. B: Similarity
plot and bootscan analysis for Changchun077. C: Similarity and bootscan analysis for Changchun103 (others not shown). A window size of 500
nucleotides in increments of 20 nucleotides at a time was used. Positions containing gaps were not excluded from the comparison.
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genome sequences were closely related to EV71 subtype B, while
VP1 sequences were closely related to EV71 subtype C. The
incongruent phylogenetic relationships suggest that the recombi-
nation event has occurred.
Many reports have described recombination in enteroviruses as
a general phenomenon between different types of enteroviruses of
the same species [8,16,17,18,19] Further analyses of the full-length
genomic sequences of the seven isolates by similarity plot and
bootscan analysis showed that genetic recombination events
existed between CA4, CA5, CA16, EV71B and EV71C strains
(Fig. 2). The 59UTR region of the seven Changchun strains were
unclassified due to a low sequence homology to any reference
strain. The 3D region of the seven strains had the highest similarity
to CA16 (Fig. 3). Most of the structural protein (P1) region
resembled that of the prototype EV71 type C. The non-structural
protein domains (P2 and P3) showed a high degree of similarity
with CA4, CA5 and EV71 type B in different regions.
Recombination appeared to be frequent in the P2 and P3 regions
but rare in the P1 region, probably because of the structural
constraint that exists to properly assemble the viral capsid. The
non-structural regions, P2 and P3, are likely the hot spots for
recombination in enteroviruses. Our present findings are consis-
tent with a previous study demonstrating, through genomic
analysis, that several recombination breakpoints were located
within the P2 and P3 regions of enteroviruses of the same species,
while no breakpoints were found within the P1 regions. However,
the seven Changchun strains consistently clustered together in all
of the analyzed regions, suggesting all these EV71 sequences had a
single common ancestor, and kept circulating and evolving within
Moreover, genetic recombination of these seven Changchun
strains was similar to the SHENZHEN-98 strain first classified as
subtype C4 in mainland China by bootscan analysis (data not
shown). Genetic recombination could result in the emergence of
viruses with altered pathogenic potential, which could cause a
serious public health threat for children because inventing a
vaccine for a rapidly mutating virus is very difficult. There is an
urgent need to establish an effective HEV71 surveillance system to
tackle the increasing threat that these viruses pose in China.
Phylogenetic analysis on full-length or partial sequences from
isolates obtained from severe or mild disease patients in
Changchun did not reveal any clustering with EV71 viruses
circulating in China (Fig. 5). Li et al  described several sites
important for virulence that are not shown in these seven
Changchun strains from fatal and non-fatal cases. Additionally,
there was no relationship between the genome sequences from
severe or mild patients and fatality. Further studies will be required
to identify viral determinants of severe HFMD in Northeast
Materials and Methods
This study has obtained ethics approval from the ethics
committee at the First Hospital of Jilin University. Written
informed consent was obtained from the parents of all the children
involved in our study.
Information of seven EV71 patients
Seven EV71 strains were chosen from 84 throat swab samples
obtained from HFMD patients in 2010; the EV71 strains were
identified with real-time PCR. Of the seven EV71 strains, the
Changchun011 and Changchun128 strains were from patients
with mild symptoms, such as slight fever, oral ulcers and skin
rashes on their palms and soles; Changchun014 and Chang-
chun103 strains were from patients who died; Changchun063
strains were from patients with severe complications that survived;
Changchun072 and Changchun077 strains were from patients
who exhibited typical clinical symptoms of CNS involvement, such
as fever, vomiting and myoclonic jerking. All the patients were
hospitalized at the First Hospital of Jilin University.
Reverse transcription PCR
Viral RNA was extracted from 200 ml of throat swabs using
TRIzol (Invitrogen). The cDNA was generated using the High-
Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
and oligo-dT primers according to the supplier’s instructions. Nine
pairs of overlapping primers were designed according to the
conserved regions of the Anhui fuyang, Chongqing1, Henan1,
Shenzhen98 strains. The PCR parameters for all the primer pairs
were as follows: cDNA were denatured at 94uC for 4 min. The
amplification was performed in 35 cycles consisting of a
denaturing step for 30 s at 94uC, a primer annealing step for
30 s at 50uC to 56uC, and a two-part elongation step for 1 to
2 min at 72uC, then extended at 72uC for 5 to 8 min. The
reactions were analyzed by electrophoresis on 1.0% agarose gels.
Amplifications were either sequenced directly or purified with
an E.Z.N.A.Gel Extraction Kit (OMEGA), cloned into the
pGEM-T Easy vector (Promega, USA) and sequenced with T7
and SP6 primers. All sequencing was performed by Sangon
Biotech (Shanghai Co., Ltd.) using the BigDyeterminatorv3.1 kit
and ABI-PRISM3730XL DNA sequencer (Applied Biosystems,
The EV71 full-length genomes were acquired by assembling all
of the fragments using the DNAMAN5.2.2 software.
The alignments of the seven Changchun EV71 strains and
reference sequences were achieved with the MEGA4 program and
Clustal W software. Phylogenetic and molecular evolutionary
analyses were conducted using the neighbor-joining method and
Kimura 2-parameter model with 1000 bootstraps pseudorepli-
cated with the MEGA5 program [15,21]. Bootstrap values lower
than 70% were hidden. The length of nucleotides used for the
analysis varied, depending on the purpose of the particular
analysis, which is clearly indicated in the Results section.
To analyze bootscanning and nucleotide similarity between
Changchun EV71 and other HEV genomes, the sequence
alignments were first completed with MEGA4.1. The results were
then analyzed using bootscan analysis in SimPlot, version 3.5.1.
The neighbor-joining method and Kimura 2-parameter model
Figure 4. Phylogenetic analysis for partial genomes from the Changchun strains. A: Based on the entire P1 gene region of the Changchun
strains, B: Based on the P2 region of the Changchun strains, C: Based on the 2A region of the Changchun strains, D: Based on the 3C region of the
Changchun strains The & icon indicates the fatal cases; m indicates the severe cases;Nindicates the mild cases.
Characterization of Enterovirus 71
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Characterization of Enterovirus 71
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were selected for all bootscanning . The window and step sizes Download full-text
were determined based on the intent of the analysis and the length
of the sequences. The reference sequences were CA2, 3, 4, 5, 6, 7,
8, 10, 12, 14, and 16 prototype strains, the poliovirus1 strain,
human enterovirus 68, 69, 76, 89, 90, 91, and 92 prototype strains,
the EV71 A prototype strain, BrCr, the B4 genotype representa-
tive strain, AM396587-UH1PM1997 and C1 genotype represen-
tative strain DQ341359 -SAR-98.
We are grateful to Juan Du for excellent technical assistance and useful
Conceived and designed the experiments: XFY WZ XW. Performed the
experiments: XW CZ. Analyzed the data: WZ XFY XW KZ. Contributed
reagents/materials/analysis tools: WZ JN KZ. Wrote the paper: WZ XFY
XW. Obtained clinical samples: WB JN. Sequence analysis: XW CZ.
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Figure 5. Phylogenetic analysis of seven Changchun strains and GenBank database sequences of the EV71 strains from other
provinces in China. A: Based on the complete genome sequence, B: Based on the VP1 region sequence, C: Based on the 59-UTR region sequence, D:
Based on the P2 region sequence,.. The & icon indicates the fatal cases; m indicates the severe cases;Nindicates the mild cases.
Characterization of Enterovirus 71
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