Phylogenetic analysis of the VP1 gene of Enterovirus 71in Guangzhou during the high occurrence period of 2008

Ying-xian Yin • Zhi-ying Ou • Yi Xu •

Rong Zhou • Hui-min Xia

Received: 22 September 2013 / Accepted: 27 January 2014 / Published online: 11 February 2014

� Springer Science+Business Media New York 2014

Abstract An outbreak of hand, foot, and mouth disease

(HFMD) in Guangzhou in 2008 affected over 10,000

children and resulted in high hospital admission rates. To

investigate the molecular epidemiological pattern of EV71

infections in Guangzhou, throat swab samples were col-

lected from 102 children clinically diagnosed with HFMD

from May to July of 2008 in Guangzhou. Partial VP1 (virus

protein 1) fragments of Enterovirus 71 (EV71) isolates

were sequenced, and used alongside EV71 sequences

entered in GenBank to construct a phylogenetic tree using

MEGA5.0. Blast and phylogenetic analyses showed that all

21 sequences belonged to subgenogroup C4 of EV71. In

early May, diverse strains were circulating in Guangzhou,

but by July, only a small number of these strains could be

detected. These results could indicate that geographic and

climatic features may affect the epidemic characteristics of

EV71, and that some C4 strains might retain their infec-

tivity at higher temperatures.

Keywords Enterovirus 71 � VP1 gene � Phylogenetic

analysis � Molecular epidemiology

Introduction

Human enterovirus 71 (EV71) is a major causative agent of

hand, foot, and mouth disease (HFMD), and compared with

other types of Coxsackie virus A16, more frequently causes

complications of the central nervous system. The rapid

identification and classification of enteroviruses is needed

for monitoring outbreaks of HFMD. Molecular diagnostic

methods based on reverse-transcriptase polymerase chain

reaction (RT-PCR) and sequencing techniques have

become the new gold standard for HFMD diagnosis,

offering greater sensitivity and specificity than traditional

methods [1–4].

The genomic RNA of Enteroviruses comprises a single

open reading frame (ORF) encoding a polyprotein (pro-

ducing P1, P2, and P3) flanked by nontranslated regions

(NTR) at the 50 and 30 ends. The 50-NTR has been the most

frequent target for RT-PCR detection, and for enterovirus

classification [5]. Further typing of individual serotypes

into serogroups and subgroups based on the 50-NTR is

problematic as it is highly conserved within serotypes. VP1

offers an attractive alternative to the 50-NTR as it contains

major antigenic epitopes, and has been found to be highly

variable among enteroviruses. In addition, high correla-

tions between VP1 nucleotide sequences and serotyping, as

determined by neutralization tests, have been documented,

indicating that the VP1 gene could be used as a determinant

for typing of human enteroviruses. This molecular dis-

tinction between serotypes is very useful for the typing and

classification of enterovirus isolates in the clinical labora-

tory [6, 7].

EV71 has been reported in many countries and regions,

particularly the Western Pacific Region [8–11]. During the

first six months of 2008, outbreaks of HFMD were reported

across several provinces in China. The initial cases of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11262-014-1046-z) contains supplementarymaterial, which is available to authorized users.

Y. Yin (&) � Z. Ou � Y. Xu � H. Xia

Institute of Pediatrics, Guangzhou Women & Children’s Medical

Center, Guangzhou 510623, China

e-mail: yinyingxian@gmail.com

R. Zhou

Guangzhou Institute of Respiratory Diseases,

Guangzhou 510230, China

123

Virus Genes (2014) 48:538–542

DOI 10.1007/s11262-014-1046-z

HFMD caused by EV71 infection were reported in Fuyang,

Anhui Province, East China. This was followed by a high

occurrence of HFMD in Guangzhou, Guangdong Province,

South China. In the current report, we studied the molec-

ular epidemiology of EV71 infection among young chil-

dren in Guangzhou during the high occurrence period of

2008, via analyzing samples from 102 pediatric patients at

the Guangzhou Children’s Hospital with clinical HFMD,

and who were suspected as enterovirus infection between

May 2008 and July 2008.

VP1 sequences of 50 EV71 strains obtained from Gen-

Bank were included in this analysis, allowing the genera-

tion of dendrograms containing 71 EV71 strains. These

strains were isolated from the United States, Japan, Tai-

wan, Malaysia, Singapore, mainland China, Bulgaria,

Hungary, and the United Kingdom. Homology searches

were carried out using the BLAST program [http://www.

ncbi.nlm.nih.gov/BLAST]. Multiple-sequence alignments

were performed with CLUSTALW [http://www.ebi.ac.uk/

Tools/clustalw2/index.html] [12].

The alignment outputs were converted to MEGA format,

and phylogenetic analyses conducted using MEGA version

5.0 [13]. Dendrograms were constructed using the neighbor-

joining (NJ) and maximum parsimony (MP) algorithms in

the MEGA program. Statistical significance of phylogenies

was estimated by bootstrap analysis with 1,000 pseudore-

plicates. Nucleotide sequences GZCH01–GZCH21 newly

obtained in this study have been submitted to GenBank and

accession numbers assigned from FJ377563–FJ377583

(Table S1).

Total RNA was extracted from soaking solution of

throat swabs using TRIZOL (Invitrogen). RT-PCR was

successfully performed using EV71 specific primers (50-TCT GTC ACC CTT GTA ATA CCA TGG AT-30, 50-TAR GCR CTC GCR GGT GAC ATG AA-30; R = A, G),

generating a DNA product of 834 bp, visualized on a 2 %

agarose gel. The 834-bp fragment was observed for 21 of

the 102 (20.6 %) specimens obtained from clinically

diagnosed HFMD cases (Fig. S1). Of these 21 samples, 13

were collected in May, two in June, and four in July (Table

S1). BLAST-n was used to align these 21 sequences with

EV71 sequences extracted from GenBank. The alignment

data showed that all 21 sequences belonged to EV71. With

the exception of GZCH11, which was almost similar to

EV71/GDFS/3/2008, all the remaining sequences shared

the greatest sequence homology with isolates from Fuyang,

Anhui Province. The phylogenetic relationship—between

42 EV71 strains, including worldwide reference strains,

and the 21 clinical samples used in this study—was

deduced by the NJ method based on the partial VP1 coding

region (nucleotide position 2,438–2,947). The worldwide

reference strains were representative of most strains of

genogroups A, B, and C. BrCr was the single prototype of

genogroup A. Strains belonging to genogroups B and C had

been in circulation in recent years in various parts of the

world. Phylogenetic analysis of the 21 clinical samples and

typical EV71 isolates was based on the alignment of partial

VP1 gene sequences (510 nucleotides), and the construc-

tion of phylogenetic trees using the NJ method. CA16-G10

was used as the outgroup. The phylogenetic tree shows that

all 21 sequences belong to subgenogroup C4 (Fig. S2). The

high bootstrap value (99 %) for the 21 clinical samples

with Shzh04-12 and Shzh04-38 supports that they should

be clustered into the C4 subgenogroup. Major lineage of

genogroup and subgenogroup in this tree is apparent with

high bootstrap values, which is in accordance with pub-

lished data [3, 14, 15].

To further reveal the phylogenetic relatedness among

these 21 clinical samples and other isolates of subgeno-

group C4, particularly those from mainland China, another

tree was constructed using NJ and MP methods (Fig. 1). In

this analysis, EV71 shzh98 (AF302996), the first isolate

reported in mainland China and belonging to subgenogroup

C4, was used as the outgroup. The two trees have similar

topologic structure. GZCH11 has a high bootstrap value

with EV71/GDFS/3/2008 (99 %), an isolate from Guang-

dong Province during the same period of 2008. The

remaining 20 clinical samples clustered into a clade with

other isolates from mainland China, supported by a boot-

strap value of 70 %. In the current study, phylogenetic

analysis demonstrated that epidemic strains of EV71 iso-

lated in Guangzhou could be grouped into subgenogroup

C4. GZCH11 (FJ377573) was most closely related to

EV71/GDFS/3/2008 and an outlier of the C4 subgeno-

group, but the high bootstrap value (91 %) strongly sug-

gests that it should be included in the C4 subgenogroup

(Fig. 1). GZCH12, GZCH14, GZCH09, GZCH03,

GZCH01, GZCH08, and GZCH19 form a single clade with

an 83 % bootstrap. GZCH13, GZCH15, and GZCH16

cluster as a single clade with GZCH04, and are more

closely related to strains Zhuhai-171 and Zhuhai-152 than

to other strains.

HFMD caused by EV71 has been reported sporadically

in many parts of mainland China since 1998 [15]. Localized

outbreaks of HFMD at geographically distinct locations

occur annually in China, but before 2008, simultaneous

outbreak of HFMD across several provinces in one season

had not been reported. In 2008, isolates of EV71 from

several provinces had been sent to the Center for Disease

Control and Prevention of China for further study. How-

ever, little is understood about the relationship between the

2008 outbreak in Guangzhou and other epidemics across

China. Sequencing of the 50-NTR region of samples of

children clinically diagnosed with HFMD in the early out-

break of 2008, among whom 81.4 %(83/102) were positive

for 50-NTR amplification, showed some samples strongly

Virus Genes (2014) 48:538–542 539

123

identified with Fuyang/17.08/3, an isolate from Fuyang,

Anhui Province (data not shown). To further understand the

phylogenetic relationship of EV71 strains isolated from two

distant regions, the current study used partial VP1 sequen-

ces from 21 samples to construct phylogenetic trees with

reference sequences stored in GenBank.

According to phylogenetic analysis based on VP1

sequences, EV71 is currently divided into three genogroups:

A, B, and C [16]. BrCr prototype strain is the only member of

genogroup A. Most EV71 isolates belong to either geno-

group B or C, and they are further classified into five sub-

genogroups: B1–B5 and C1–C5 [9–11, 14, 17]. C4 first

reported as Shzh98 (AF302996) was isolated in Shenzhen,

China in 1998. It was debated whether the outbreaks in

mainland China during 2008 were linked to the 2004 C4

outbreaks in Taiwan. The two methods used in the

Fig. 1 Dendrograms constructed using the neighbor-joining (a) and

maximum parsimony (b) methods showing the genetic relationships

among 21 clinical EV71 strains collected from Guangzhou Children’s

Hospital, and reference sequences of genogroup C retrieved from

GenBank. The alignments were based on partial VP1 gene sequences.

Branch lengths are proportional to the number of nucleotide

differences. The bootstrap values in 1,000 pseudoreplicates for major

lineages within the tree are shown as percentages. Values less than

70 % were not shown. The marker denotes a measurement of relative

phylogenetic distance. Shzh98 belonging to C4 subgenogroup was

used as the outgroup in this analysis. The date in parenthesis tells only

the date of samples collected, not the date of strains. The different

months of samples collected were colored (green for May, orange for

June, and red for July) (Color figure online)

540 Virus Genes (2014) 48:538–542

123

construction of phylogenetic trees in the current study show

similar topology (Fig. 1). Despite some differences in the

tree twigs, the evolutionary hierarchy is almost identical.

Shzh98 at the tree roots can be considered as the ancestor of

the C4 subgenogroup. GZCH11 and EV71/GDFS/3/2008,

which are near the root, were isolated from two adjacent

regions of Guangdong Province.

It is of interest to note that for nearly ten years, all isolates

from mainland China belonged to subgenogroup C4, and that

their genotypes were not affected by epidemic situations in

adjacent regions including Japan, Taiwan, Singapore, and

Malaysia [15, 18–20]. In addition, neither genogroup A nor

B has been reported in mainland China since 2001. This

contrasts with Taiwan, where there was a more complex

epidemic pattern of EV71, where subgenogroups B1, B4, B5,

C2, C4, and C5 had been simultaneously predominant over

the last 10 years [17, 21–24]. These results indicate that

geographic and climatic features could affect the epidemic

characteristics of EV71, and specific subgenogroups might

be sustained in mainland China.

An interesting phenomenon encountered in this study

was the presence of multiple strains of EV71 cocirculating

during the early stages of outbreak compared with fewer

strains detected in clinical samples during the later stages

of outbreak. Our analysis showed a great diversity of EV71

strains belonging to subgenogroup C4 circulating in Gu-

angzhou in early May. Many of these isolates shared

similar identities to Zhuhai-213, Fuyang22, and Zhe-

jiang08, or with ancestral strains (according to the phylo-

genetic tree) C4, such as Shzh04-12. In contrast, in late

July, only isolates from the same branch of the phyloge-

netic tree as Fuyang/17.08/3 were detected in the Gu-

angzhou area. Isolates GZCH01, GZCH03, GZCH05,

GZCH06, GZCH07, GZCH08, GZCH09, GZCH10,

GZCH11, GZCH14, GZCH18, GZCH19, and GZCH21

were collected in early May 2008, while GZCH02,

GZCH04, GZCH17, and GZCH20 were collected in late

July (Fig. 1). GZCH02, GZCH04, GZCH13, GZCH15,

GZCH16, and GZCH20 clustered in a clade within the C4

subgenogroup, and were phylogenetically related to Zhu-

hai152 and Zhuhai171.

Many factors such as temperature, humidity, and

population mobility can affect the epidemiological pattern

of EV71; however, when environmental conditions are

kept the same for all isolates, theoretically, the differential

pattern for different isolates should be predominantly

attributed to genetic factors. Mutations in specific regions

of viral genomic RNA can alter virus sensitivity to tem-

perature [25]. Our previous sequencing results also

showed that no nucleotide mutations in position 251 of

the 3D polymerase region of all isolates obtained were

found. Other mutations besides this region might alter

sensitivity to temperature, while this task is time

consuming and needs a large number of samples to verify.

Based on the phylogenetic tree, it could be surmised that

from May to July, with a gradual rise in temperature in

Guangzhou, the higher temperature is optimal for isolates.

Urashima et al. argued the number of patients with

HFMD will increase if summer and autumn temperatures

increase [26]. An epidemiological study in Guangzhou

showed that each 1 �C rise in temperature corresponded

to an increase of 9�38 % in the weekly number of HFMD

cases [27]. Our findings seem to contradict their conclu-

sions, although those authors argue that their viewpoint is

based on seasonal models. The average temperature in

Guangzhou in June and July was higher than that in May.

Perhaps more importantly, the history climatological data

indicate that there were two temperature peaks greater

than 35 �C in July, one in June, and nil in May (Fig. S3).

In late July (the hottest time in Guangzhou) versus early

May, incidences of HFMD have decreased dramatically

(Table S3). In our study, the number of EV71 cases was

reduced in late July, as was the mean diversity of C4

subgenogroups of EV71(Fig. S3).

Other factors such as relative humidity, atmospheric

pressure, and wind velocity may also affect the epide-

miological pattern of HFMD; nevertheless, the most

likely climatological factor that affects the genetic

diversity should be temperature, because other factors

except temperature only contribute to the viability and the

spread of viruses, and influence of which is fair for all

isolates. Another driving force accelerating the evolution

of virus is host–pathogen interaction. However, this study

is hard to be carried out in very inadequate samples and

population. Moreover, in the first outbreak of EV71

infection, virus–host interaction including immune state is

relatively simpler than other enteroviruses, and so EV71

was utilized to study the temperature–genetic diversity in

our study.

Further studies on the role of temperature in virus sus-

ceptibility for different isolates of subgenogroup C4 will be

undertaken using cell culture and site-directed mutagenesis

of EV71 genes. Viability test in high temperature greater

than 35 �C for different isolates should be performed too.

Moreover, a greater number of isolates should be included

for more extensive epidemiologic studies.

Acknowledgments Financial support was provided by grants from

the Guangzhou Municipal Health Bureau (30307-2080048) and the

Guangdong Natural Science Foundation (s2012010008319).

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