Cp

QQK2

ARRAA

KECP

1

aw2eiwpmd2

dmwrar

0h

Journal of Virological Methods 187 (2013) 127– 131

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

jou rn al h om epage: www.elsev ier .com/ locate / jv i romet

haracterization of enterovirus 71 capsids using subunit protein-specificolyclonal antibodies

ingwei Liu, Xulin Huang, Zhiqiang Ku, Ting Wang, Fei Liu, Yicun Cai, Dapeng Li,ibin Leng, Zhong Huang ∗

ey Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 411 Hefei Road, Shanghai00025, China

rticle history:eceived 31 March 2012eceived in revised form 13 August 2012ccepted 20 September 2012vailable online 6 October 2012

eywords:nterovirus 71

a b s t r a c t

Enterovirus 71 (EV71), a member of the Enterovirus genus of the Picornaviridae family, is one of themajor causative agents of hand-foot-and-mouth disease (HFMD), which is prevalent in the Asia-Pacificregion. In this article, a set of capsid subunit protein-specific antibodies was used to characterize theEV71 structural protein processing and to determine the composition and assembly of EV71 capsids.SDS-PAGE and Western blot analyses showed that the capsids of a purified EV71 preparation, whichlacked viral infectivity, were composed of processed VP0, VP1 and VP3, all of which co-assembled intoparticles. Analyses of infectious EV71-containing cell lysate revealed the presence of VP2, in addition

apsidolyclonal antibody

to VP0, VP1 and VP3, suggesting that the cleavage of VP0 into VP2 and VP4 is important for infectivity.Immunofluorescent staining with the three specific antibodies demonstrated that the capsid subunitproteins co-localized in the cytoplasm of cells infected with EV71. The results add new information onthe processing, assembly and localization of EV71 capsid proteins, and demonstrate the usefulness ofthe capsid protein-specific antibodies for virological investigation and for development of vaccines anddiagnostic reagents.

. Introduction

Enterovirus 71 (EV71) is the major causative agent of hand-foot-nd-mouth disease (HFMD) (Lee et al., 2009; Wong et al., 2010),hich is prevalent currently in the Asia-Pacific region (Wong et al.,

010; Xu et al., 2010), China in particular (Xu et al., 2012; Yangt al., 2011; Zhang et al., 2009, 2010). EV71 infection occurs mainlyn children under the age of five. A portion of patients infected

ith EV71 develop severe neurological and cardiopulmonary com-lications, such as polio-like paralysis, brain stem encephalitis,eningitis, and pulmonary edema, which may ultimately lead to

eath (reviewed in Lee et al., 2009; McMinn, 2002; Wong et al.,010).

EV71 is a member of the Enterovirus genus of the Picornaviri-ae family. Its genome is a single-stranded, positive sense RNAolecule of ∼7410 bases, and encodes a single large polyproteinhich can be divided into structural P1 and nonstructural P2 and P3

egions (reviewed in McMinn, 2002). In many enteroviruses, suchs poliovirus and foot-and-mouth disease virus, the structural P1egion is processed by viral proteinase to yield the subunit proteins

∗ Corresponding author. Tel.: +86 21 54653077; fax: +86 21 63843571.E-mail address: huangzhong@sibs.ac.cn (Z. Huang).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2012.09.024

© 2012 Elsevier B.V. All rights reserved.

VP0, VP1 and VP3, all of which co-assemble to form empty cap-sids (Fry et al., 2005; Hellen and Wimmer, 1992); further cleavageof VP0 into VP2 and VP4 by an autocatalytic mechanism involv-ing the encapsidating RNA is required for generation of infectiousmature virions (Basavappa et al., 1994; Curry et al., 1997; Hellenand Wimmer, 1992; Hindiyeh et al., 1999). However, whether thisis the case for EV71 has not been investigated sufficiently, due partlyto the lack of suitable immunological reagents to identify subunitprotein species.

In this study, a set of capsid subunit protein-specific antibodieswas used to characterize the EV71 structural protein processingand to determine the composition and assembly of EV71 capsids.In addition, the localization of capsid proteins within infected cellswas investigated by immunofluorescent staining.

2. Materials and methods

2.1. Cells and viruses

RD and Vero cells were grown in DMEM (Gibco, Grand Island,

NY, USA) supplemented with 10% FBS, 100 unit/ml penicillin and100 �g/ml streptomycin) at 37 ◦C with 5% CO2. EV71 strain G081(genotype C4) was propagated in RD cells. Virus titers was deter-mined by the microtitration method using RD cells and were

1 gical M

eaw

2

vatewTtEt(oCf(5aptp(5(bFptwwde1

2

bwowfjw(L

2

1B0a

2

wwW

28 Q. Liu et al. / Journal of Virolo

xpressed as the 50% Tissue Culture Infectious Dose (TCID50)ccording to the Reed–Muench method (Reed, 1938). Purified EV71as obtained from Hualan Inc. (Henan, China).

.2. Capsid subunit protein-specific polyclonal antibodies

The anti-VP0 guinea pig polyclonal antibody was described pre-iously (Feng et al., 2011). The anti-VP3 guinea pig polyclonalntibody was generated by immunization of guinea pigs withhe recombinant VP3 protein of Coxsackievirus A16 (CVA16) (Liut al., 2011b), this antibody was found to cross-react stronglyith EV71 (data not shown) and hence was used in this study.

he anti-VP1 polyclonal antibody was generated by immuniza-ion of rabbit with recombinant EV71 VP1 protein produced from. coli. To construct the EV71 VP1 prokaryotic expressed vec-or, RNA was extracted from EV71-infected RD cells using TrizolInvitrogen, Carlsbad, CA, USA), and then reverse transcribed usingligo(dT) primer and M-MLV reverse transcriptase (Invitrogen,arlsbad, CA, USA) according to Manufacturer’s instructions. VP1ragment was amplified from the resultant cDNA with primersforward 5′-GGTCCATGGGAGATAGGGTGGCAGATG-3′ and reverse′-GCGCTCGAGAAGAGTGGTGATCGCTGAG-3′), digested with NcoInd XhoI, and then inserted into prokaryotic expression vectorET26b from the same sites, giving rise to pET26-VP1. Prokaryo-ic expression of pET26-VP1 and purification of recombinant VP1rotein were carried out following protocols described previouslyLiu et al., 2011b). One rabbit was immunized subcutaneously with0 �g of VP1 protein in the presence of complete Freund’s adjuvantSigma, St. Louis, MO, USA) to generate VP1-specific antibody, andoosted twice with the same amount of antigen plus incompletereund’s adjuvant (Sigma, St. Louis, MO, USA) at 3 and 6 weeks post-riming, respectively. At two weeks after the last booster injection,he animal was killed and sera collected. This animal experimentas approved by the SJTU Ethics Committee for Animal Care andas carried out in the SJTU animal facility. Specific IgG titers wereetermined by endpoint titer ELISA as described previously (Liut al., 2011b) except that the 96-well ELISA plates were coated with00 ng/well of the recombinant EV71 VP1 protein.

.3. SDS-PAGE and Western blotting

Protein samples were mixed with SDS-PAGE sample buffer,oiled and then separated on 12% polyacrylamide gels. Proteinsere either visualized by Coomassie blue staining or transferred

nto PVDF membranes for Western blot analysis. Membranesere probed with a capsid subunit protein-specific antiserum,

ollowed by a corresponding horseradish peroxidase (HRP) con-ugated secondary antibody. Positive signals on the membranes

ere developed by chemiluminescence using the BeyoECL Plus kitCat# P0018, Beyoime, Shanghai, China), recorded using a LAS-4000uminescent Image Analyzer (Fujifilm Life Science).

.4. Sucrose gradient analysis

Protein samples (purified EV71 or cell lysates) were layered onto0–50% sucrose gradients and subjected to ultracentrifugation in aeckman SW60Ti rotor at 156,000 × g for 3 h at 4 ◦C. Ten fractions of.4 ml each were taken from top to bottom and subjected to ELISAnd/or Western blot analyses.

.5. Indirect ELISA

Each sucrose gradient fraction (10 �l/well) was added into 96-ell microtiter plates and incubated at 37 ◦C for 2 h, followed byashing three times with PBST (PBS containing 0.05% Tween-20).ells were blocked with 200 �l PBST plus 5% milk at 37 ◦C for

ethods 187 (2013) 127– 131

1 h, washed, then incubated with 50 �l/well of the guinea pig anti-VP0 antisera diluted (1:2000) in PBST plus 1% milk at 37 ◦C for 2 h.After washing, wells were incubated with 50 �l/well of goat anti-guinea pig conjugated to horseradish peroxidase diluted (1:5000)in PBST plus 1% milk at 37 ◦C for 1 h. For color development, wellswere washed then 50 �l/well of TMB mixture was added and incu-bated for 5–10 min, followed by 50 �l/well of 1 N H3PO4 to stop thereaction. Absorbance was measured at 450 nm in a 96-well platereader.

2.6. Electron microscopy

Purified EV71 preparations were stained negatively with 0.5%aqueous uranyl acetate, and transmission electron microscopy wascarried out with a Philips CM-12S microscope.

2.7. Immunofluorescent staining

Vero cells infected with EV71 were fixed with 4% paraformalde-hyde for 20 min, followed by treatment with 1% NP40 diluted inPBS for 10 min at room temperature. The fixed cells were incu-bated with 10% normal goat serum for 30 min, then with a rabbit orguinea pig primary antibody for 30 min, followed by a correspond-ing secondary antibody for 30 min. The secondary antibodies usedincluded FITC-conjugated (Santa Cruz Biotechnology, Santa Cruz,CA, USA) or Alexa Fluor 488-conjugated (Cat# A11073, Invitro-gen) anti-guinea pig IgG for emitting green fluorescence, and Texasred-conjugated (Santa Cruz Biotechnology) or Alexa Fluor 555-conjugated (Cat# A21429, Invitrogen) anti-rabbit IgG for emittingred fluorescence. At last, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min. All incubation steps were carriedout at 37 ◦C and the cells were rinsed four times with PBS betweensteps. The stained cells were examined using a conventional epi-fluorescent microscope (Leica, Wetzlar, Germany) or a confocalmicroscope (Leica).

2.8. Confocal microscopy

Confocal microscopy was carried out using a Leica SP5 confocallaser scanning microscope. In general, the following parameterswere used, pinhole aperture: 1 P AU; UV laser (405 nm) inten-sity: 12.5%; Ar laser (488 nm) intensity: 15%; He–Ne laser (543 nm)intensity: 36%; scan speed: 200 Hz. Images in 1024 × 1024 formatwere acquired in the DAPI, Alexa Fluor 488 and Alexa Fluor 555channels, and processed using the Leica LAS AF version 2.6.0 soft-ware.

3. Results

3.1. Protein composition of EV71 capsids

To determine the protein composition of EV71 capsids, we firstanalyze a purified EV71 preparation by SDS-PAGE analysis. Threemajor Coomassie blue-stained bands with molecular weight ofapproximately 38-, 34-, and 26-kDa, respectively, were evident(Fig. 1A, lane 1). Detection with capsid subunit protein-specificpolyclonal antibodies revealed that, the 38-kDa band reacted onlywith the anti-VP0 polyclonal antibody (Fig. 1A, lane 2), the 34-kDaband with the anti-VP1 (Fig. 1A, lane 3), and the 26-kDa band withthe anti-VP3 (Fig. 1A, lane 4). These results indicated that the 38-,34- and 26-kDa bands represent VP0, VP1 and VP3 subunit pro-teins, respectively. To analyze further the EV71 capsid composition,

lysate from cells infected with EV71 was subjected to Westernblotting. As expected, positive signals of ∼34 and ∼26 kDa weresolely detected by the anti-VP1 and anti-VP3, respectively (Fig. 1B,lanes 2 and 3). However, two bands, one of ∼38 kDa and another of

Q. Liu et al. / Journal of Virological Methods 187 (2013) 127– 131 129

F estera emb4 with

∼sdbt

3

asibpbwgIwra

typ#to

3

bEbbmEsi

tflnSfVi

4. Discussion

In this study, the composition and assembly of EV71 capsidswere investigated using a set of capsid subunit protein-specific

Fig. 2. Co-assembly of capsid subunit proteins into particles. Purified EV71 sam-

ig. 1. Determination of protein composition of EV71 capsids. (A) SDS-PAGE and Wnd subsequently stained with Coomassie blue dye (lane 1), or transferred to PVDF m). Lane M, protein marker. (B) Western blotting analyses of lysates of cells infected

28 kDa, reacted with the anti-VP0 antibody (Fig. 1B, lane 1). Theize of the latter is as predicted for VP2, which is a cleavage producterived from VP0 and hence can cross-react with the anti-VP0 anti-ody. Thus, the expected molecular weight and its reactivity withhe anti-VP0 indicates that the ∼28 kDa band represents the VP2.

.2. VP0, VP1 and VP3 co-assemble into particles

To determine whether the structural subunit proteins co-ssemble to form capsids, purified EV71 was sedimented on 10–50%ucrose gradients and the resulting gradient fractions were exam-ned for the presence of subunit proteins by ELISA and Westernlotting. Detection with anti-VP0 showed that VP0 accumulatedredominantly in the fraction #6 in the ELISA (Fig. 2A). Westernlotting with the three antibodies demonstrated VP0, VP1 and VP3ere concentrated in the fraction #6 (Fig. 2B–D), strongly sug-

esting that they co-existed and assembled in a particulate form.ndeed, the presence of spherical particles of ∼30 nm (Fig. 2E)

as revealed by transmission electron microscopy. Together, theseesults indicated that VP0, VP1 and VP3 subunit proteins co-ssembled to form EV71 capsids.

The cell lysate from cells infected with EV71 was also subjectedo sucrose gradient sedimentation followed by Western blot anal-sis with VP0-, VP1-, or VP3-specific antibodies. As shown in Fig. 3,ositive signals were mainly detected in the fractions #5, #6 and7 for all three antibodies, with the highest intensity in the frac-

ion #6. In addition, the profile of VP2 was found to resemble thatf VP0, but with less intensity (Fig. 3A).

.3. Intracellular localization of capsid subunit proteins

The localization of the capsid subunit proteins was determinedy immunofluorescent staining of Vero cells infected with EV71.pifluorescence microscopy showed that all three polyclonal anti-odies positively stained EV71-infected cells (Fig. 4A, D and G),ut not mock-infected ones (data not shown), whereas the preim-une serum (as the control) failed to stain cells infected with

V71 (Fig. 4J). This result indicated that the three capsid protein-pecific antibodies were able to recognize specifically EV71 by themmunofluorescent staining assay (IFA).

The fluorescence patterns of stained cells were examined fur-her by confocal microscopy. For all three primary antibodies,

uorescence appeared to localize in the cytoplasm (Fig. 5A–L), butot in the nucleus (stained by DAPI), of the cells infected with EV71.imultaneous staining with pairs of two primary antibodies raisedrom two different animal species revealed tight colocalization ofP1 with VP0/VP2 (Fig. 5M–P), and of VP1 with VP3 (Fig. 5Q–T). It

s therefore concluded that VP1, VP0/VP2, and VP3 colocalize.

n blot analyses of purified EV71. Purified EV71 virus was separated by SDS-PAGE,rane for Western blotting with anti-VP0 (lane 2), anti-VP1 (lane 3) or anti-VP3 (lane

EV71 with anti-VP0 (lane 1), anti-VP1 (lane 2) or anti-VP3 (lane 3).

ple was layered onto 10–50% sucrose gradients and subjected to centrifugation asdescribed in Section 2. Ten fractions (0.4-ml each) were taken from top to bottomand subjected to biochemical and biophysical analyses. (A) Profile of reactivity ofgradient fractions with the anti-VP0 analyzed by ELISA. Data are mean ± SD of trip-licate samples from each fraction. (B) Western blot of fractions with anti-VP0. (C)Western blot of fractions with anti-VP1. (D) Western blot of fractions with anti-VP3.(E) Transmission electron microscopy of purified EV71. Bar = 100 nm.

130 Q. Liu et al. / Journal of Virological Methods 187 (2013) 127– 131

Fig. 3. Sucrose gradient analysis of unpurified, infectious EV71 virus. Crude lysatesof EV71-infected Vero cells were heated at 56 ◦C for 30 min to inactivate the virus,layered onto 10–50% sucrose gradients and then subjected to centrifugation asdescribed in Section 2. Ten fractions (0.4-ml each) taken from top to bottom weresr

aaepeVo

Fig. 4. Epifluorescence microscopy. EV71-infected Vero cells, which had been fixedas described in Section 2, were incubated with one of the primary antibodies, includ-ing the rabbit anti-VP1 (A–C), the guinea pig anti-VP0 (D–F), the guinea pig anti-VP3(G-I), and a guinea pig preimmune serum (J–L). The cells were subsequently incu-

FaA

ubjected to Western blotting with (A) anti-VP0, (B) anti-VP1, and (C) anti-VP3,espectively.

ntibodies. The results demonstrate that co-expression of VP0, VP1nd VP3 is sufficient for EV71 capsid formation. Interestingly, West-rn blot analysis of crude EV71-infected cell lysate revealed the

resence of VP2, in addition to VP0, VP1 and VP3 (Fig. 1B). The gen-ration of VP2 is likely due to the autocatalytic cleavage of VP0 intoP2 and VP4, which has been shown to be essential for productionf infectious virions of some enteroviruses, such as poliovirus and

bated with a corresponding secondary antibody (either Texas Red-conjugated goatanti-rabbit IgG antibody emitting red fluorescence or FITC-conjugated goat anti-guinea pig IgG antibody emitting green fluorescence), and also stained briefly withDAPI (B, E, H, and K). The resulting samples were examined under an epifluorescencemicroscope.

ig. 5. Intracellular localization of EV71 capsid proteins revealed by confocal microscopy. EV71-infected Vero cells were fixed, stained with one or two primary antibodiess indicated, followed by incubation with a corresponding secondary antibody (either Alexa-555-conjugated goat anti-rabbit IgG antibody emitting red fluorescence orlexa-488-conjugated goat anti-guinea pig IgG antibody emitting green fluorescence). The stained cells were examined under a Leica confocal microscope.

gical M

f1tcwwbEtpia2g

cdipswvcateip

pdbv

A

t(gg

Q. Liu et al. / Journal of Virolo

oot-and-mouth disease virus (Basavappa et al., 1994; Curry et al.,997). Indeed, the purified, VP2-lacking EV71 preparation used inhis study was found to be non-infectious as it could not induceytopathic effects even after three blind passages (data not shown),hereas the unpurified EV71 in the lysate was highly infectiousith a titer of ∼108 TCID50/ml. Similar observation has recently

een made by Liu et al. (2011a). They reported that the emptyV71 particles purified by sucrose gradient zonal ultracentrifuga-ion were of low viral infectivity and composed of three capsidroteins (VP0, VP1 and VP3), and the solid particles with high viral

nfectivity and RNA content contained four proteins (VP1, VP2, VP3nd VP4). Together, the results from this study and others (Liu et al.,011a) support the hypothesis that cleavage of VP0 is critical to theeneration of infectious EV71 virions.

The utility of the polyclonal antibodies against recombinantapsid proteins in detection and characterization of EV71 wasemonstrated by ELISA (Fig. 2A), Western blot (Figs. 1–3) and

mmunofluorescent staining (Figs. 4 and 5). The use of subunitrotein-specific antibodies allowed clear determination of thepecie and integrity of each individual capsid component, whichould be important for quality control of EV71 inactivated virus

accines currently in clinical trials. In addition, the availability ofapsid subunit protein-specific antibodies raised from differentnimal species (rabbit and guinea pig) permitted the first study ofhe colocalization of the individual EV71 capsid subunits. Undoubt-dly, these antibodies can also facilitate the investigation of thenteraction/colocalization between EV71 capsid proteins with hostroteins.

In conclusion, the results add new information on therocessing, assembly and localization of EV71 capsid proteins, andemonstrate the usefulness of the capsid protein-specific anti-odies for virological investigation as well as for development ofaccines and diagnostic reagents.

cknowledgements

The authors thank Dr. Wenqi An of Hualan Inc. for providing

he purified EV71 samples. This work was supported by a grant#KSCX2-YW-BR-2) from the Chinese Academy of Sciences. Z.H.ratefully acknowledges the support of SA-SIBS scholarship pro-ram.

ethods 187 (2013) 127– 131 131

References

Basavappa, R., Syed, R., Flore, O., Icenogle, J.P., Filman, D.J., Hogle, J.M., 1994. Role andmechanism of the maturation cleavage of VP0 in poliovirus assembly: structureof the empty capsid assembly intermediate at 2.9 A resolution. Protein Sci. 3,1651–1669.

Curry, S., Fry, E., Blakemore, W., Abu-Ghazaleh, R., Jackson, T., King, A., Lea, S., New-man, J., Stuart, D., 1997. Dissecting the roles of VP0 cleavage and RNA packagingin picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus. J. Virol. 71, 9743–9752.

Feng, Y.F., Liu, Q.W., Ku, Z.Q., Wen, J.J., Shan, H., Huang, Z., 2011. Expression of VP0protein of enterovirus 71 in Escherichia coli and generation of the correspondingpolyclonal antibodies in guinea pigs. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 27,535–538.

Fry, E.E., Stuart, D.I., Rowlands, D.J., 2005. The structure of foot-and-mouth diseasevirus. Curr. Top. Microbiol. Immunol. 288, 71–101.

Hellen, C.U., Wimmer, E., 1992. Maturation of poliovirus capsid proteins. Virology187, 391–397.

Hindiyeh, M., Li, Q.H., Basavappa, R., Hogle, J.M., Chow, M., 1999. Poliovirus mutantsat histidine 195 of VP2 do not cleave VP0 into VP2 and VP4. J. Virol. 73,9072–9079.

Lee, T.C., Guo, H.R., Su, H.J., Yang, Y.C., Chang, H.L., Chen, K.T., 2009. Diseases causedby enterovirus 71 infection. Pediatr. Infect. Dis. J. 28, 904–910.

Liu, C.C., Guo, M.S., Lin, F.H., Hsiao, K.N., Chang, K.H., Chou, A.H., Wang, Y.C., Chen, Y.C.,Yang, C.S., Chong, P.C., 2011a. Purification and characterization of enterovirus71 viral particles produced from vero cells grown in a serum-free microcarrierbioreactor system. PLoS One 6, e20005.

Liu, Q., Ku, Z., Cai, Y., Sun, B., Leng, Q., Huang, Z., 2011b. Detection, characteriza-tion and quantitation of coxsackievirus A16 using polyclonal antibodies againstrecombinant capsid subunit proteins. J. Virol. Methods 173, 115–120.

McMinn, P.C., 2002. An overview of the evolution of enterovirus 71 and its clinicaland public health significance. FEMS Microbiol. Rev. 26, 91–107.

Reed, L.J.M.H., 1938. A simple method of estimating 50 percent endpoints. Am. J.Hyg. 27, 493–499.

Wong, S.S., Yip, C.C., Lau, S.K., Yuen, K.Y., 2010. Human enterovirus 71 and hand, footand mouth disease. Epidemiol. Infect. 138, 1071–1089.

Xu, J., Qian, Y., Wang, S., Serrano, J.M., Li, W., Huang, Z., Lu, S., 2010. EV71: an emerginginfectious disease vaccine target in the Far East? Vaccine 28, 3516–3521.

Xu, W., Liu, C.F., Yan, L., Li, J.J., Wang, L.J., Qi, Y., Cheng, R.B., Xiong, X.Y., 2012. Dis-tribution of enteroviruses in hospitalized children with hand, foot and mouthdisease and relationship between pathogens and nervous system complications.Virol. J. 9, 8.

Yang, F., Zhang, T., Hu, Y., Wang, X., Du, J., Li, Y., Sun, S., Sun, X., Li, Z., Jin, Q., 2011.Survey of enterovirus infections from hand, foot and mouth disease outbreak inChina, 2009. Virol. J. 8, 508.

Zhang, Y., Tan, X.J., Wang, H.Y., Yan, D.M., Zhu, S.L., Wang, D.Y., Ji, F., Wang, X.J., Gao,Y.J., Chen, L., An, H.Q., Li, D.X., Wang, S.W., Xu, A.Q., Wang, Z.J., Xu, W.B., 2009.An outbreak of hand, foot, and mouth disease associated with subgenotype C4of human enterovirus 71 in Shandong, China. J. Clin. Virol. 44, 262–267.

Zhang, Y., Zhu, Z., Yang, W., Ren, J., Tan, X., Wang, Y., Mao, N., Xu, S., Zhu, S., Cui,A., Zhang, Y., Yan, D., Li, Q., Dong, X., Zhang, J., Zhao, Y., Wan, J., Feng, Z., Sun,J., Wang, S., Li, D., Xu, W., 2010. An emerging recombinant human enterovirus71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyangcity of China. Virol. J. 7, 94.