Microbiol Immunol 2015; 59: 477482

doi: 10.1111/1348-0421.12281

ORIGINAL ARTICLE

MA104 Cell line presents characteristics suitable for

enterovirus A71 isolation and proliferation
Zhi-Hui Li1, Ying-Ying Yue1, Peng Li1, Nan-Nan Song1, Bingqing Li1, Ying Zhang2, Hong Meng1,
Guo-Sheng Jiang1 and Lizeng Qin1
1

Department of Microbiology, Institute of Basic Medicine, Shandong Academy of Medical Sciences, Key Laboratory of Rare and
Uncommon Diseases, 18877 Jingshi Road, Jinan 250062 and 2Jinan Infectious Disease Hospital, 22029 Jingshi Road, Jinan 250021,
China

ABSTRACT
Enterovirus A71 (EV-A71), one of the most important causative agents of hand, foot and mouth
disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection
spectrum of EV-A71 in different cell lines remains unknown. Therefore, in this study, the biological
characteristics of EV-A71 Subgroup C4 in different cell lines were investigated. To this end, the
infectivity of EV-A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different
host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for
EV-A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of
cell lines can be infected by EV-A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD,
MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50) values calculated in
viral proliferations (ranged from 107.6 to 107.8); the TCID50 being negatively associated with the time
to appearance of CPE. Proliferation curves demonstrated that EV-A71Jinan1002 amplies more
efciently in MA104, Hep-2 and RD cells. Remarkably, the virus isolation rate was much higher in
MA104 cells than in RD cells. Thus this study, to our knowledge, is for the rst to explore the infection
spectrum of EV-A71 subgroup C4 in such a large number of different cell lines. Our data provide
useful reference data for facilitating further study of EV-A71.
Key words

enterovirus A71 (EV-A71), host cells, biological characteristics.

Enterovirus A71 is one of the major pathogens that

causes HFMD in children, symptoms including convulsion, ataxia, brain-stem encephalitis and poliomyelitislike paralysis (1, 2). Several outbreaks have been
reported since EV-A71 was identied in 1969, including
in Australia, Japan, Brazil, Malaysia, Singapore, Taiwan,
and recently in mainland China (3, 4). Several
diagnostic methods, such as RT-PCR and ELISA, that
have been developed for viral research and clinical
studies (58) have enabled accurate identication of
EV-A71 infection, including identifying the subgroup of
EV-A71.

Obviously, these research tools play important roles in

detection and diagnosis of EV-A71 infection. Nevertheless, isolation of EV-A71 is still the gold standard for
denitive diagnosis of HFMD. To date, researchers have
used a variety of cell lines to investigate EV-A71,
including RD, HT29, Hep-2 and Vero cell lines (9, 10).
Although, RD cells have reportedly been successfully
employed to isolate EV-A71 from clinical specimens, the
isolation rates are generally low, the highest reported
isolation rate being 66.7% from stool specimens cultured
for 3 days in RD cells (11), which is much lower than
the RT-PCR positive rate of 91.3% (12, 13). Although,

Correspondence
Lizeng Qin, Director, Department of Microbiology, Shandong Academy of Medical Sciences, Key Laboratory of Rare and Uncommon Diseases, 18877
Jingshi Road, Jinan 250062, China. Tel: 86 0531 82919701; fax: 86 0531 82919930; email: lizeng_qin@163.com.
Received 31 March 2015; revised 9 June 2015; accepted 28 June 2015.
List of Abbreviations: CPE, cytopathic effect; CT, threshold cycle value; EV-A71, enterovirus A71; HFMD, hand, foot and mouth disease;
SCARB2, scavenger receptor class B, member 2; TCID50, 50% tissue culture infective dose.

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477

L. Qin et al.

RT-PCR or ELISA are the primary means of studying

EV-A71 infection, these techniques are inadequate for
investigation of further characteristics, such as receptor
screening and pathogenic mechanisms (1416). Therefore, identifying suitable cell lines for EV-A71 isolation
for further experiments and investigating this virus's
biological features in different cell lines is extremely
useful. Thus far, the spectrum of host cells that are
susceptible to infection remains largely unclear; little is
known about EV-A71 isolation despite the fact that
MA104 cells have been demonstrated to be suitable
for rotavirus isolation (17). In the present study,
we attempted to infect 18 different cell lines with the
EV-A71Jinan1002 strain, which had been isolated
from children with HFMD in Jinan city, mainland
China. Our aim was to clarify the host cell spectrum
and the biological characteristics in these cells. We
showed, for the rst time, that the MA104 cell line can be
used for EV-A71 isolation, as can several other cell lines,
some of which achieved comparably high proliferation.
Thus, our data provide useful reference data for
facilitating further study of EV-A71.

MATERIALS AND METHODS

Cell culture
RD, HEK-293, Hep-2, SGC-7901, Caco-2, MCF-7, A549,
SMMC-7721, HEL, HeLa, Marc145, MA104, MDBK, P3
and ST were cultured in RPMI-1640 (Invitrogen,
Carlsbad, CA, USA) supplemented with 10% heatedinactivated FBS (HyClone, Logan, UT, USA), 100 IU
penicillin/mL and 100 mg streptomycin/mL at 37C in a
humidied atmosphere under 5% CO2.
Virus isolation
Collection of human samples was approved by the
Research Ethical Committee of Jinan Infectious Diseases
Hospital (Jinan, China). Prior to collection of fecal
samples, all parents or caregivers of the eligible children
were asked to provide written informed consent and
given explanations of the study. Thirty-eight fecal
samples that had been obtained from children with
HFMD in Jinan Infectious Disease Hospital and stored at
80C were used. Briey, when RD or MA104 cells (18)
had formed a monolayer, fecal sample supernatants
diluted with RPMI-1640 (Invitrogen) supplemented
with 10% heated-inactivated FBS (HyClone), 100 IU
penicillin/mL and 100 mg streptomycin/mL were added,
after which sterilization was achieved by ltration
through a 0.22 mm membrane lter. The cells were
then placed at 37 in a 5% CO2 environment and the CPE
observed daily for 7 days.
478

EV-A71 virus (TCID50) assay

TCID50 assays were performed following the method of
Reed and Muench (19). Briey, when they had formed a
monolayer (104 cells per well) in 96-well cell culture
plates (Corning, Cambridge, MA, USA), cells were
inoculated with 100 uL tenfold serial dilutions of virus
stock previously isolated in RD cells. Ten wells were
used for each dilution and incubated for 5 days, during
which the cultures were checked for the presence of
CPE at 4 hr intervals. The experiments were repeated in
triplicate.
EV-A71 infection
EV-A71Jinan1002 virus isolated previously and stored at
80C was used. Briey, 18 different passage cell lines
were added separately to 24-well cell culture plates.
When the cells had formed a monolayer, the supernatant
was aspirated and the virus diluted with RPMI-1640
supplemented with 2% FBS, 100 IU penicillin/mL and
100 mg streptomycin/mL (maintenance buffer). The cells
were then infected with EV-A71Jinan1002 at MOI of
5 (TCID50 tested in RD) and placed at 37C in a 5% CO2
environment for 2 hr. Unattached viruses were washed
out with RPMI-1640. Maintenance buffer (500 uL/well)
was added to the cell culture plates. If no CPE had
occurred after 5 days, observation was continued for
another 7 days.
RT-PCR
Viral RNA was extracted from the infected cells using an
RNA Kit (Tiangen, Beijing, China). cDNA was prepared
from total RNA using AMV reverse transcriptase
(Thermo Scientic, Rockford, IL, USA) with Oligo(dT)
as primer. Briey, 20 mL reaction mixtures were
incubated for 60 min at 42C and 10 min at 72C in
succession, and then maintained at 4. PCR was
performed with 1 mL of reverse transcribed product,
12.5 mL of HotStart Taq MasterMix (Tiangen) and 1 mL
of each primer (71S: 50 -GCAGCCCAAAAGAACTTCAC-30 ; 71A: 50 -ATTTCAGCAGCTTGGAGTGC-30 ) in
25 mL of reaction mixture. The reactions were performed
at one cycle of 95C for 5 min, followed by 32 cycles of
95C for 30 s and 52C for 30 s and another 72C for 30 s
for extension. Three independent experiments were
conducted for each sample. PCR products were visualized by agarose gel electrophoresis.
Real-time RT-PCR
cDNA was prepared as described above. Quantitative
RT-PCR was performed using an Applied Biosystems
7500 Sequence Detection system (Applied Biosystems,
2015 The Societies and Wiley Publishing Asia Pty Ltd

EV-A71 in MA104 cells

Foster, CA, USA), with 1 mL of RT-PCR product,

12.5 mL of SYBR Green/ROX qPCR Master Mix
(Thermo Scientic), and 0.05 mL of diluted ROX in
25 mL of reaction mixture. The reactions were performed
at one cycle of 95C for 10 min, followed by 40 cycles of
95C for 15 s and 60C for 1 min in 96-well plates.
All reactions were run in triplicate. Threshold cycle
values were determined using default threshold settings
and the mean CT determined from duplicate PCRs.
EV-A71 RNA assay
Real-time RT-PCR for detection of EV-A71 was
performed as described above. A12, a plasmid encoded
complete cDNA of EV-A71, a gift from the Academy of
Military Medical Sciences (Beijing, China) was used as a
standard and the standard curve used to quantify the virus.
Statistic analysis
Statistic analyses were performed with the x2 and Student's
t-test using SPSS software (version 13.0; Chicago, IL,
USA). P < 0.05 was considered statistically signicant.

RESULTS
EV-A71 isolation rate is higher in MA104
than in RD cells
Firstly, the rates of isolation of EV-A71 from RD and
MA104 cells were compared, using 38 fecal specimens
obtained from patients infected with EV-A71. The virus
isolation rate from MA104 cells was 63.2% (24/38),
whereas for RD cells it was only 36.8% (14/38), which
is signicantly lower than for MA104 cells (P < 0.05,
x2 test).
EV-A71Jinan1002 can be grown in a broad
spectrum of cell lines
Having demonstrated the difference between MA104 and
RD cell lines in virus isolation, we were intrigued to
determine the size of the host cell infection spectrum.
First, experiments to screen the 18 host cell lines for their
suitability for EV-A71Jinan1002 strain infection were
performed. It was found that, EV-A71Jinan1002 did not
infect cell lines derived from lower species of animals,
such as cattle (MDBK, P3), dogs (MDCK), swine (ST,
PK15) and mice (Hepa1-6), but did infect all cell lines
derived either from humans or monkeys (Table 1). To
conrm infections of all cells used in this study, 5 days
after EV-A71Jinan1002 inoculation, total RNAs were
extracted and RT-PCR performed. Of the 18 cell lines
investigated, 12 did have the EV-A71 genome, conrming
infection with EV-A71Jinan1002. CPE was evidenced
2015 The Societies and Wiley Publishing Asia Pty Ltd

mainly as cell shrinkage, rounding, cytoplasmic vacuolar

changes and fragmentation (Fig. 1).
TCID50s of EV-A71 in different cell lines
To further characterize the infected cell lines, TCID50 for
EV-A71 in 12 cell lines were calculated. It was found that
TCID50 from HEK293, RD, MA104 and Marc145 cells
were comparably high, ranged from 107.6 to 107.8
(Table 1). Additionally, there were lower TCID50 values
from tumor cells (P < 0.05, Student's t-test). Moreover,
among all the cell lines, CPE appeared earliest in MA104
(about 16 hr after virus infection), whereas it appeared
last in SMMC-7721 (about 46 hr) (Table 1).Interestingly,
except for HeLa cells, it was found that the earlier the CPE
appeared, the higher the TCID50 values (Fig. 2).
Amplication of EV-A71Jinan1002 varies in
different cell lines
To investigate the replication kinetics of virus proliferation, a standard curve was established using A12 plasmid
as standard. Concentrations were measured with
NanoDrop2000 (Thermo Fisher, MA, USA). Two hours
following virus inoculation, cells were washed once with
PBS. At 2, 4, 8, 19, 24 and 32 hr post-inoculation, total
RNA was extracted. A proliferation curve for EV-A71
was constructed using values obtained by qPCR to
conrm the viral infection spectrum and times of
appearance of CPE. It was found that the detected viral

Table 1. TCID50 and the time of appearance of CPE

Origin

Cell line

logTCID50

x  Sd

Average time of appearance

of CPE (hr)

Human

RD
HEK293
Hep-2
SGC-7901
Caco-2
MCF-7
A549
SMMC-7721
HEL
HeLa
Marc145
MA104
Hepa1-6
MDBK
P3
ST
PK15
MDCK

7.7  0.3
7.8  0.4
5.5  0.2
5.6  0.1
3.3  0.1
4.7  0.2
3.3  0.1
3.8  0.1
6.3  0.2
3.4  0.1
7.6  0.3
7.8  0.3

17.3
20
25.3
25.3
41.3
26.7
37.3
46.7
21.3
18.7
17.3
16

Monkey
Mouse
Bovine
Porcine
Canine

Note: The times shown for appearance of CPE are the average from
three sets of observations.

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L. Qin et al.

Fig. 1. CPE varies between different cell lines

after infection with EV-A71 (200). Six cell lines
(RD, HEK-293, SMMC-7721, HeLa, HEL and
MA104) infected by EV-A71Jinan1002 display
typical CPE as described in the Results section. Hep2, SGC-7901, Caco-2, MCF-7, A549 and Marc145
showed similar CPE (data not shown).

cDNA copies varied largely between cell lines, indicating

different infectivities for different cell lines. The
proliferation curve showed clearly that EV-A71 proliferates more efciently in MA104 and RD cells (Fig. 3).

DISCUSSION
EV-A71, which is among the most common viruses that
are very specic for humans, only infects humans,
especially children under 5 years old. However, many
animals, such as Rhesus monkeys, suckling mice, young
gerbils, and infant Tupaia belangeri Chinese have
reportedly been infected with this virus in different
laboratories (2022). In our experience, the search for
perfect cell lines to further study EV-A71 infection in
vitro is ongoing. In this study, we demonstrated that
MA104 is suitable for EV-A71 proliferation. We
investigated a broad spectrum of 18 cell lines of human,
non-human primate or small animal origin for EV-A71
480

infection. Additionally, we ascertained the kinetics of

EV-A71 proliferation by constructing a proliferation
curve from values obtained by RT-qPCR.
Many factors, such as receptors and the intra- and
extra-cellular environment, can affect the infectivity of
viruses. Cell receptors have been considered to be the
primary determinant of tissue tropism. Although we did
not design experiments to detect receptors used on
different cell lines in the present study, we demonstrated
that EV-A71Jinan1002 can proliferate in all tested cell
lines of human and monkey origin. This result is in good
agreement with the fact that cell lines from humans and
monkeys are rich in expression of receptors that enable
EV-A71 to enter into host cells (23). Study of EV-A71
infection has shown that SCARB2 receptor is inhibited
both by anti-SCARB2 antibodies and by soluble
SCARB2, suggesting that SCARB2 plays a critical role
in the EV-A71 infection pathway (23). However, it has
become apparent that receptors are more widely
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EV-A71 in MA104 cells

Fig. 2. Correlation between TCID50 and the time of appearance

of CPE. To analyze the relationship between TCID50 and the time of
appearance of CPE caused by EV-A71Jinan1002, a curve was
constructed with version SPSS13.0 software. TCID50 is negatively
associated with the time of appearance of CPE (R2 0.566,
P 0.005, the formula: y 0.1362  9.1056).

distributed in host cells than in virus replication

sites (24). Another study has been demonstrated
that EV-A71 infection is inhibited when cells are
treated with drugs that block acidication of the
endosome (25), indicating that virus tropism may
be determined by factors other than virus receptors (26).
To determine the virulence of EV-A71 infection in vitro,
in this study, we also found that the TCID50s of EVA71Jinan1002 in MA104, HEK-293, RD and Marc145
were quite high (range from 107.6107.8). Early appearance
of viral CPE was correlated with higher TCID50s,
particularly in MA104 cells (16 hr). The detailed
mechanisms remain unclear. Studies have shown that
intracellular environments inuence many processes of
EV-A71 infection, such as type I interferons (27, 28),
melanoma differentiation-associated protein 5 (29), EVA71 3C protease (30), and, more recently reported,
miRNA (31, 32). A review by Lin et al. (26) conrms that
difference in intracellular environments inuence many
processes of virus infection, including the speed of virus
replication, spread of infection within and between tissues
or organs and development of a disease. In general, viral
proliferation is inuenced by multiple factors stemming
from both host and virus per se; the detailed mechanisms
remain largely unknown. Our ongoing project is focusing
on dening factors relating to EV-A71 virulence in MA104
cell infection. Taken together, in the current study, we have
presented characteristics of the MA104 cell line pertaining
to its suitability for EV-A71 proliferation, thus providing
useful reference data to facilitate further study of this virus.

ACKNOWLEDGMENTS

Fig. 3. Replication kinetics of EV-A71Jinan1002. The copies of

EV-A71 genome replication in six different cell lines were observed to
record the kinetics of EV-A71Jinan1002 proliferation. A12, a plasmid
encoded complete cDNA of EV-A71, was employed to set up a
standard curve. An amplication plot showed an efciency of
91.014%. A metal curve reected a high specicity. Standard
curve (R(2) 1). The proliferation curves demonstrate the viral cDNA
copies of EV-A71 and show that viral proliferation was greatest in
MA104 cells.

2015 The Societies and Wiley Publishing Asia Pty Ltd

The authors would like to thank Cheng-Feng Qin

(Academy of Military Medical Sciences, Beijing, China)
for offering the plasmid A12. This study was supported
by the National Natural Science Funds of China (Grant
No.81000720), Intramural Institutional of Basic Medicine grants (JCSY201402 and JCSY201403), a Shandong
Academy of Medical Sciences grant (2014-11), the
Department of Health and Family-plan Bureau
(2014WS0068), Shandong Provincial College of Traditional Chinese Medicine Antiviral Collaborative Innovation Center (XTCX2014), the National Science And
Technology Major Special Project (2014ZX09509001001008), China, and the Natural Science Foundation
of Shandong Province (ZR2009DQ025, ZR2010HQ039,
ZR2011HQ030 and ZR2013HQ039).

DISCLOSURE
The authors declare that they have no competing
interests.
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L. Qin et al.

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