Etiology, pathogenesis, antivirals and vaccines of hand, foot, and mouth disease

17 Pages·2015·8 MB·English

REVIEW NationalScienceReview
2:268–284,2015
doi:10.1093/nsr/nwv038
Advanceaccesspublication6July2015
MICROBIOLOGY
SpecialTopic:InfectionandImmunity
Etiology, pathogenesis, antivirals and vaccines of hand,
foot, and mouth disease
D
o
XiaoboLei1,ShengCui1,ZhendongZhao1,∗ andJianweiWang1,2,∗ wn
lo
a
d
e
d
fro
ABSTRACT m
h
Hand,foot,andmouthdisease(HFMD),causedbyenteroviruses,isasyndromecharacterizedbyfever ttps
withvesiculareruptionsmainlyontheskinofthehands,feet,andoralcavity.HFMDprimarilyaffects ://a
c
a
infantsandyoungchildren.Althoughinfectionisusuallyself-limited,severeneurologicalcomplicationsin d
e
thecentralnervoussystemcanpresentinsomecases,whichcanleadtodeath.Widespreadinfectionof m
ic
HFMDacrosstheAsia-PacificregionoverthepasttwodecadeshasmadeHFMDamajorpublichealth .o
u
challenge,rankingfirstamongthecategoryCnotifiablecommunicablediseasesinChinaeveryyearsince p.c
o
2008.ThisreviewsummarizesourunderstandingofHFMD,focusingontheetiologyandpathogenesisof m
thedisease,aswellasonprogresstowardantiviralsandvaccines.Thereviewalsodiscussestheimplications /ns
ofthesestudiesastheyrelatetothecontrolandpreventionofthedisease. r/a
rtic
Keywords: le
1MOHKeyLaboratory hand,footandmouthdisease,enterovirus,etiology,tropisms,virus–hostinteraction, -a
b
ofSystemsBiologyof pathogenesis,innateimmunity,antivirals,vaccine stra
Pathogens,Instituteof c
PathogenBiology, INTRODUCTION [7,8]. HFMD emerged in the 1970s in Japan [9], t/2/3
ChineseAcademyof /2
fromwhichpointonnumerousoutbreaksoccurred 6
MPeekdinicgaUlnSicoinenMceesdiacnadl Hilyaanffde,cftosoitn,faanntdsamnoducthhilddirseenasyeou(nHgFeMrtDha)np5riymeaarrs- intheAsia-Pacificregion[2,10–16].Afterthe1990s, 8/213
largeoutbreaksthatinvolvedmorethan10000cases 0
College,Beijing old and displays a wide range of clinical manifes- 3
6
100730,Chinaand tations [1]. The disease is characterized by fever alloccurredinthisregion[2,11,16,17](Fig.1).The 2 b
2Collaborative alongwithvesiculareruptionsmainlyontheskinof reasonsunderlyingtheseoutbreaksarestillnotfully y g
InnovationCenterfor hands,feet,andtheoralcavity.HFMDisgenerally understood. ue
s
Diagnosisand self-limited.However,severeneurologicalmanifes- In 2008 and 2009, large outbreaks of HFMD t o
Treatmentof tationscanpresentinsomecases,rangingfromasep- emerged in mainland China [2]. These outbreaks n 1
InfectiousDiseases, tic meningitis to acute flaccid paralysis and brain- madeHFMDagreatpublichealthconcerninChina, 6 N
Hangzhou310003, leadingtoitsclassificationasacategoryCcommuni- ov
China stemencephalitis(BE),whichcanbepermanently cablediseaseintheChineseNationalcommunicable em
disabledorfatal[2].TheBEcausedbyHFMDcan b
e
leadtoseverepulmonaryedemaandshock,which diseasesurveillancesystemin2008[18].From2008 r 2
∗Corresponding can induce failure of respiratory and circulatory to2014,morethan1millionHFMDcaseshavebeen 018
authors.Email: systems[3]. reportedinChinaeachyear,accordingtodataob-
[email protected]; tainedfromtheChineseNationalNotifiableDisease
HFMDwasfirstidentifiedinNewZealandand
[email protected] ReportingSystem.
Canadain1957[4].Thediseasewasdesignatedas
GreatprogresshasbeenmadeinHFMDresearch
‘hand,foot,andmouthdisease’afterasimilarout-
overthepasttwodecades,whichhashelpedtopave
Received10April break occurred in USA in 1959 [5]. HFMD reap-
thewaytowardpreventionandcontrolofHFMD.
2015;Revised13 peared in New Zealand, the UK, and in the USA
Here,wesummarizetheunderstandingofHFMD
June2015;Accepted in the 1960s [5,6]. Outbreaks of HFMD also oc-
etiologyandreviewresearchprogressonthepatho-
19June2015 curredinBulgariain1975andinHungaryin1978,
genesis, antivirals, and vaccines of enterovirus 71
and 44 and 47 deaths were recorded, respectively
(EV71),themaincausativeagentofsevereHFMD.
(cid:3)CTheAuthor(s)2015.PublishedbyOxfordUniversityPressonbehalfofChinaSciencePublishing&MediaLtd.Allrightsreserved.ForPermissions,pleaseemail:
[email protected]
REVIEW
Leietal. 269
Finland
2008
Canada
1957 UK
1959, 1963, 1966, 1994, 2014
Hungary Bulgaria
1978 1975
USA Spain
Mainland China Japan D
1959, 1963,2011-2012 2011 1981, 1983, 1986, 1973, 1978, ow
2008-2014, 2013 1983-2003,2011 n
Taiwan China loa
Th2a0il1a2nd2005V,i e2t0n1a1m-2012 1998, 2004-2009 ded
Singapore Malaysia Brunei fro
1981,2000,2008 1997 2006 m
h
ttp
s
Australia ://a
1991, 1999 ca
d
e
m
New Zealand ic
1957, 1961,1963 .o
u
p
.c
o
m
/n
s
r/a
Figure1.WorldwideepidemicsofHFMD(countryandyear).RedindicatesoutbreaksofHFMDinvolvedmorethan10000casesandblueindicates rtic
coxsackievirusA6(CV-A6)infection,whichisoneofthemostcommonpathogensofHFMDinrecentyears. le-a
b
s
Wealsodiscusstheimplicationsofthesestudiesas EVs, the Enterovirus genus is proposed to be di- tra
c
theyrelatetocontrolandpreventionofthedisease. videdintosevenspecies,includingEV-AtoDand t/2
rhinovirusA–CbytheInternationalCommitteeon /3/2
6
TaxonomyofViruses[19]. 8
ETIOLOGYOFHFMD /2
Improvement of virus detection methodology 1
3
HFMDiscausedbyEVs,whichbelongtotheEn- and disease surveillance has led to a better under- 03
6
terovirusgenusofthefamilyPicornaviridae.TheEV standingoftheetiologyofHFMD.Atleast23EV 2 b
genuscontainsmanywell-knownviruses,including serotypes,whichbelongtotwodifferentEVspecies, y g
u
polioviruses (PV), Coxsackie viruses (CV-A and - havebeenreportedtocauseHFMDoverthepast e
s
B), and ECHO viruses (E). The EV genome is a 50 years (Table 1). Among them, EV71 and CV- t o
n
single positive-stranded RNA molecule, which en- A16 are the most prevalent. CV-A16, isolated in 1
6
codesa5(cid:4)-untranslatedregion(5(cid:4)-UTR),apolypro- 1958,wasthefirstidentifiedHFMDpathogen[4], N
o
tein,anda3(cid:4)UTR.Thepolyproteinconsistsofthree followed by EV71, which was isolated in the USA ve
m
regions:P1,P2,andP3,whichareinturncleaved in1969[20].Sincethen,EV71outbreakshaveoc- b
e
intofourviralcapsidproteins(VP1–VP4)andseven curred periodically throughout the world, includ- r 2
0
non-structuralproteinsinvolvedinproteinprocess- ing in Japan [9], Bulgaria [7], Hungary [8], Aus- 1
8
ing and genome replication (2A–2C, 3A–3D) by tralia [21], Malaysia [22], UK [23], and Vietnam
EV’s proteases 2A and 3C [19] (Fig. 2). The vi- [24].CV-A16outbreaksalsooccurredinAustralia,
ral capsid is an icosahedron, composed of VP1– England,Singapore,andthemainlandChina[25].
VP4.VP1,VP2,andVP3formadeepcanyonthat CV-A16infectionandEV71infectionemergedal-
servesasthereceptor-bindingsiteatthesurfaceof ternately and these vectorshave becomethe most
the capsid. Proteins 2A and 3C are not only es- relevant pathogens of HFMD worldwide to date
sential for EV replication, but also play important [2,11,13,14]. In mainland China, HFMD was first
rolesinvirus–hostinteractions.Theprotein3Disan reported in Shanghai in 1981 [18]. Outbreaks of
RNA-dependent RNA polymerase (RdRp), which CV-A16infectionwereidentifiedinTianjinin1983
lacksproofreadingactivity,leadingtofrequentmu- and1986.EV71wasfirstisolatedinWuhanin1995
tations during EV replication. Based on the phy- [18]. The outbreaks of EV71 infection occurred
logenetics of VP1, the major antigenic protein of in Linyi, Shandong Province in 2007 and Fuyang,
REVIEW
270 NatlSciRev,2015,Vol.2,No.3
D
o
w
n
lo
a
d
e
d
fro
m
h
Figure2.Diagramoftheenterovirusgenome.Top:schematicoftheEVRNAgenome.Bottom:processingpatternofEVpolyprotein. ttp
s
://a
c
Table1.EnterovirusserotypesrelatetoHFMD. Likewise, EV71 is classified into subtypes A, B, C, a
d
e
andD.SubtypeAcontainsonlytheprototypestrain m
Species Serotypes ic
BrCr.SubtypesBandCareeachcomposedofdis- .o
EnterovirusA CV-A2,CV-A4,CV-A5,CV-A6,CV-A7,CV-A8,CV-A10,CV-A12, tinct subgenogroups (B1–B5 and C1–C5, respec- up
CV-A16,EV-A71 tively) [38]. Subtype D is represented by a single .co
m
EnterovirusB CV-B1,CV-B2,CV-B3,CV-B4,CV-B5,CV-A9,E-4,E-7,E-9,E-11,E-25, strainwhichhasbeenisolatedfromIndia[39].The /n
s
E-30,EV-B84 circulatingstrainsofEV71appeartovarywithge- r/a
Adoptedfrom[45,47,48]. ographicallocationandtime.Forexample,inTai- rticle
wan China, subgenogroups C2 and B4 were re- -a
b
AnhuiProvincein2008[26,27],initiatingtheEV71 sthpeon2s0ib0l2eefopridtehmei1c,9C984efpoirdtehmeic2,0w04h–il0e5Be4pwidaesmfiocr, strac
pandemicinmainlandChinathathaspersistedever B5 for the 2008–09 epidemic, C4 for the 2010 t/2/3
since. epidemic, and B5 for the 2011–12 epidemic [40]. /26
8
The fact that EV71 has been associated with InmainlandChinain1996,subgenogroupC2ap- /2
1
a wide spectrum of acute central nervous system peared,followedbyC3in1997.Since1998,C4has 30
3
(CNS) syndromes, including aseptic meningitis, been the dominant subgenogroup [28,41,42]. The 6
2
poliomyelitis-like paralysis, BE, and acute neuro- C4subtypesunderwentthreemajorepidemiologic b
y
genic pulmonary edema [28], makes it the pre- phases:C4bin1998–2008,C4a-1in2003–09,and gu
e
dominantserotypeinsevere(80%)orfatal(93%) C4a-2 in 2007–11 [28]. The constant changing of s
t o
laboratory-confirmed cases [2,11]. CV-A16 and subgenogroupsmakesitchallengingtodesignuni- n
1
mostotherEVserotypesmoreintendtocausemild versalvaccinesthatcoverseveralsubgenogroups. 6
N
HFMDcases[2,17,29]. In recent years, the switch of HFMD etiology o
v
RecombinationisthemajorforcethatdrivesEV has been suggested by the increased epidemics of em
b
variation [30]. CV-A16 and EV71 often circulate serotypes other than EV71 and CV-A16, includ- e
togetherandtheircoinfectionincreasesthechance ingCV-A6,CV-A10,andCV-A12[43–48].Disease r 20
1
of intertypic recombination [31,32]. A recombi- caused by a new strain of CV-A6 has been found 8
nantmayberesponsibleforthelargeHFMDout- worldwide[49–60](Fig.1),causingsevereHFMD
breaksinmainlandChina[33].Recombinationalso inchildren[61]andatypicalHFMDinadults[62–
contributes to the variation of other EV serotypes 64]. In some areas in China, CV-A6 has replaced
[34,35]. CV-A16asapredominantcausativeagent[59,65].
DuetousingRdRp,thevariationratesofEVsare TheincreasingtrendofCV-A6spreadwarrantsen-
veryhigh.Oneserotypecanbeclassifiedintosev- hancedpreciseetiologysurveillanceofHFMD.
eralsubtypesbasedonthephylogenyofVP1.CV- Together, diverse EV serotypes can cause
A16aregroupedintosubtypesAandB,withthelat- HFMD,andEV71andCV-A16arethemostcom-
terfurtherdividedintoB1a–B1candB2a–B2c[32]. mon pathogens. EV71 is the major agent causing
Although there is a difference in the definition of severecases.TheswitchofHFMDetiologyrequires
subgenogroupB1andB2,thestrainsthathavecir- apreciseEVtypinginthesurveillanceforabetter
culated in China belong to genogroup B [36,37]. HFMDcontrol.
REVIEW
Leietal. 271
PATHOGENESISOFEV71 alloftheaspectsofHFMDpathologyinhumans.For
example,inmonkeymodels,unlikeinhumans,the
Pathologicalfeatures
pathogenic process, including pulmonary edema
Most pathological research on EVs has been fo- and neuron impairment, is dependent upon infec-
cused on EV71. Studies on EV71 in HFMD pa- tion route, only presenting after intracerebral in-
tients mainly cause neurological effects by induc- oculation with EV71 rather than other infection
inginflammationintheCNS,butnotinotheror- pathways [79,81]. Furthermore, in mice, adaption
gans. The pathological features of EV71-induced increasesthevirulenceofEV71anddoesnotreflect
viralencephalitisinfatalcasesincludeneuronopha- thenaturalcellandtissuetropism[82].Pulmonary
gia,perivascularcuffing,focaledema,andneutrophil edemahasneverbeenobservedintheimmunode-
andmacrophageinfiltration[66–69].Theinflamma- ficientmousemodelinfectedbytheadaptedEV71 D
o
tiondistributesmainlytothehypothalamus,brain strains[83–85].Thehumanscavengerreceptorclass w
n
stem, spinal cord, and cerebellar dentate nucleus B, member 2 (SCARB2) transgenic mice display loa
d
alongthemotornervepathway,indicatingthatthe clinical features most similar to humans, exhibit- e
d
virusspreadsintotheCNSthroughtheretrograde ingataxia,paralysis,anddeathafterinfection.These fro
peripheralmotornerve[66,67].Autopsyexamina- micearesusceptibletoEV71clinicalisolatesatdif- m h
tions in EV71 fatal cases revealed brainstem en- ferent ages and with different inoculation routes. ttp
s
cephalomyelitis, extensive pulmonary edema, and However, pulmonary edema symptoms are absent ://a
pulmonary hemorrhages [69–71]. However, viral inthismodel,whichlimitsitsapplicationinEV71 ca
d
antigenswereonlydetectedinthebrainstemandin pathogenesisexploration[80].Despitetheseshort- em
thespinalcordbutnotinthelungtissue,suggesting comings, our knowledge has been greatly potenti- ic
.o
thatEV71-inducedpulmonaryedemaisneurogenic atedfromstudiesintheseanimalmodels. up
[69,70]. .co
m
Although limited, clinical observations provide /n
s
clues that some inflammatory mediators, includ- Receptorsandviralfactorsmodulatecell r/a
ing cytokines and chemokines, play an important andtissuetropism rtic
role in the pathogenesis of EV71-induced BE and le-a
othercomplications.Thesemediatorsincludeinter- Virus–receptorinteractionisthefirststepofavirus bs
leukin6(IL-6),IL-10,IL-13,tumornecrosisfactor life cycle, determining cell and tissue tropism and tra
c
α (TNF-α), IL-1β, IL-8, and monocyte chemoat- pathogenicity.TwohumanreceptorsforEV71and t/2
/3
tractant protein 1 [72–75]. Of these factors, ele- CV-A16wereidentifiedin2009:SCARB2[86]and /2
6
vatedIL-6mightbeaprognosticparameterforclin- P-selectionglycoproteinligand-1(PSGL-1)[87].In 8/2
ical severity [73,74]. Significantly higher levels of addition,annexinIIandsialylatedglycanshavealso 13
0
IL-10 and IL-8 in the cerebrospinal fluid of the beenreportedascandidatecoreceptorsforEV71in- 3
6
patients with encephalitis and pulmonary edema fection[88].SCARB2islocalizedinthelysosomal 2 b
y
than in uninfected controls suggests that the in- membrane and is ubiquitously expressed in many g
u
creased inflammatory cytokines in the serum of humantissuesandcelltypes,includinginneurons es
EV71 infected patients may originate in the cere- intheCNS.Itparticipatesinmembranetransporta- t o
n
brospinalfluid[72].Althoughtherearesomestud- tionandthereorganizationofendosomal/lysosomal 16
ies providing evidence that the vascular cell adhe- compartments,andshuttlesbetweenthesecompart- N
o
v
sionmolecule1andcyclooxygenases-2-inducedac- mentsandtheplasmamembrane[86,89,90].PSGL- e
m
tivation of nuclear factor κB (NF-κB) might rep- 1isexpressedexclusivelyonmyeloidandTlympho- b
e
resent the pathway for mediating the production cytesandplaysaroleintheearlystagesofinflamma- r 2
0
ofinflammatorycytokinesandchemokines,thede- tion[87,89,90]. 18
tailed mechanism of the inflammatory pathogen- In infected organs of a viral disease, cell-to-
esis induced by EV71 infection remains unclear cell spread substantially contributes to disease
[76,77]. pathogenesis. It has been reported that different
As an alternative to clinical and autopsy re- cell types use SCARB2 and PSGL-1 to mediate
sources, experimental murine and non-human pri- EV71 entry through clathrin- and caveolar-
mate models of EV71 infection have been devel- dependent endocytosis, respectively [91,92]. The
oped to explore the pathogenesis of EV71 infec- differential expression of EV71 receptors on dif-
tion. These resources include a monkey model, ferent cell types and tissues are considered the
neonate and adult immunodeficient mice-adapted primary determinants of tissue tropism [89]. For
EV71models,andanEV71receptortransgenicmice example,EV71patientspresentingwithbrainstem
model [78–80]. However, some limitations exist encephalitis, autonomic nervous system dysreg-
withthesemodels,asnoneaccuratelyrecapitulates ulation, and pulmonary edema have a high level
REVIEW
272 NatlSciRev,2015,Vol.2,No.3
of proinflammatory cytokines in serum and cere- patternspresentonthevirus,whicharerecognized
brospinal fluid. Therefore, it is speculated that the by pathogen recognition receptors (PRRs) [101].
interaction of EV71 with PSGL-1 on lymphocytes PRRs involved in the recognition of EV71 and in
mayspreadthevirusandproducetheinflammatory mediatedtypeIIFNsproductionincludemelanoma
cytokines by trafficking to the CNS to promote differentiation-associatedgene5(MDA5),retinoic
brain stem encephalitis and pulmonary edema de- acid-induciblegeneI(RIG-I),Toll-likereceptor3
velopment.Incontrast,itwassuggestedthatEV71 (TLR3),andTLR7[102,103].
uses the active retrograde axonal transport system Despitetheserobustimmuneresponses,several
toentertheCNSinanorallyinfectedEV71murine linesofevidencesuggestthatEV71hasevolvedeva-
model [93]. Together, these findings suggest that sionstrategiestoantagonizetheIFN-mediatedin-
a single receptor interacting with EV71 is not nateimmuneresponses(Fig.3).Forexample,dur- D
o
sufficient to elicit EV71 brain stem encephalitis; ing EV71 infection, type I IFNs are undetectable w
n
rather, different receptors may play different roles in cell-based systems and in animal model, while loa
ondifferentcelltypesandtissuesatdistinctstagesof IFNα/β treatment increases the survival rate of de
d
EV71infection.However,severallinesofevidence mice, and neutralizing antibody to IFNα/β exac- fro
m
suggest that SCARB2 plays a dominant role in erbatesEV71-induceddisease[104,105].Addition- h
efficient EV71 infection and the development of ally,EV71almostdoesnotstimulatetheexpression ttp
s
systemicdiseaseinhumans,whileothercandidates ofantiviralgenes,suchasIFN-stimulatedgenes54 ://a
mayactascoreceptorsorfunctiontohelpinvading (ISG54)andISG56[105]. ca
d
the target cells in vivo. First, stable expression of ThemainIFNantagonistsencodedbyEV71have em
humanSCARB2permittedreplicationofalltested been identified as two viral proteases, 2A and 3C. ic
.o
EV71andCV-A16strainsinnon-susceptiblemouse Theydirectlytargetdiversekeycytosolicmolecules up
cells,whilehumanPSGL-1onlyenablessomerep- ofthetypeIIFNsignalingpathwaystoblockhost .co
m
resentativeEV71strains[86,87].Second,SCARB2 immuneresponses.EV712Aproteasedirectlytar- /n
s
iscapableofviralbinding,viralinternalization,and getsMDA5andinducesitscleavage,whichisacom- r/a
viral uncoating, while PSGL-1 showed inability to mon event in the infection of EV species [106]. rtic
le
induce viral uncoating, resulting in low infection EV712Aprotein,butnot3C,canalsotargetthemi- -a
b
efficiency in cells expressing PSGL-1 [94]. Last, tochondriaanti-viralsignalingprotein(MAVS,also s
SCARB2 transgenic mice are susceptible to infec- namedasIPS-1,VISA,Cardif)andcleaveitatmulti- trac
tion by both EV71 clinical isolates and CV-A16, plesites[107].TheresultingEV712A-cleavedfrag- t/2
/3
displaying EV71 neurotropism, neuropathology, mentsofMAVScannotactivatetypeIIFNsproduc- /2
6
andclinicalfeatures.PSGL-1transgenicmice,onthe tion[108].Althoughthe3CofCV-B3wasreported 8/2
1
otherhand,canonlybeinfectedbyamousemuscle- to cleave overexpressed MAVS, cleavage by EV71 3
0
adaptedEV71strainbutnottheclinicalisolates[80, 3Chasnotbeenobserved[106,107].Inanalterna- 36
2
84,95,96]. tivepathway,EV713CproteasecaninhibitRIG-1- b
y
InadditiontoreceptorsofEV71,studiesondif- mediatedtypeIIFNresponsesbyimpedingthefor- g
u
e
ferentmurinemodelshaveindicatedthatviralfac- mationofafunctionalcomplexbetweenRIG-Iand s
tors,suchasviral5(cid:4) and3(cid:4) UTR,geneticmodifica- MAVS[105]. t on
tion on the VP1 region, and 3D polymerase, con- ArecentstudyshowedthatTLR3signalingac- 16
N
tribute to cell and tissue tropism as well as viral tivationinmacrophagesisimportantforprotecting o
v
pathogenesis [89,97–100]. However, the detailed older mice against EV71 infection [103]. Accord- em
mechanismofhowtheseviralfactorscontributeto ingly, our results indicated that EV71 suppresses be
thecellortissuetropismremainsunclear. TLR3-mediatedtypeIIFNresponses.Inthispro- r 2
0
1
cess, the protease 3C interacts with TIR domain- 8
containingadaptorinducingIFN-β(TRIF),anim-
portantadaptorproteinofTLR3,andcleavesitupon
Evasionofhostinnateimmuneresponses
the proteolytic activity of 3C [109]. As a strong
Duringtheviruslifecycle,EVsusemultiplefactors antagonist of IFN, 3C also directly cleaves inter-
from both viral and host for their own benefit. In feron regulatory factor 7 (IRF7), the downstream
response,thehostdevelopscorrespondingimmune molecule of RLRs and TLR3 signaling pathways
responses to prevent a viral infection. Innate im- [110].Importantly, thecleavagefragmentsdonot
munity is the first line to defense against invading limit the replication of EV71, which is consistent
pathogens, including type I interferon (IFN) pro- with their ability to inhibit IFN production. Co-
duction at the early stages and subsequent activa- incident with these observations, EV71 3C blocks
tion of downstream events. The type I IFNs pro- typeIIFNsynthesisinamousemodel[111].These
moterisactivatedbypathogen-associatedmolecular studiessuggestthat3C,asanantagonist,mayplay
REVIEW
Leietal. 273
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
s
r/a
rtic
le
-a
b
s
tra
c
t/2
/3
/2
6
8
/2
1
3
0
3
6
2
Figure3.InhibitionoftypeIIFNinductionbyEV71atdiversestepsinsignalingpathways.EV71infectionissensedbyPRRsincludingRIG-Ilikereceptors by
(RLRs)andTLRs.EV71targetsthesesignalingpathwaysbythe2A,2C,and3Cantagonists.2AcleavesMAVSandMDA5,andinducesthedegradation gu
ofIFNAR1.2CinhibitsIKKβphosphorylation.3CcleavesTRIF,IRF9,IRF7,andtheTAK1complexandalsointerruptstheinteractionbetweenRIG-Iand es
MAVS. t on
critical roles in evading innate immune responses IFN-mediatedactivationofISGsviainterferingwith 16 N
underphysiologicconditions. the Janus activated kinase (Jak)-signal transduc- ov
It is thought that NF-κB signaling is also ers and activators of transcription (STAT) signal- em
important for activating IFNs or inflammatory imngedpiaattehdwpahyso.sEphVo7r1yliantfieocntioofnSTcaAnTi1n,hSibTitAtTh2e,IJFakN1-, ber 2
cytokine production in TLR and RLR signaling 0
1
pathways. We have demonstrated that EV71 3C and tyrosine kinase 2 through inducing cleavage 8
also inhibits the activation of NF-κB by cleaving of type I IFN receptor 1 (IFNAR1) by 2A pro-
the transforming growth factor-β-activated kinase tease [115]. Besides IFNAR1, EV71 3C protease
can cleave IRF9 to block JAK1-STAT signaling
1 (TAK1) complex [112]. Furthermore, another
[116].
EV71 non-structural protein, the 2C helicase, has
beenalsodemonstratedtoinhibitTNF-α-mediated
NF-κB activation by suppressing phosphory-
lation of IκB kinase β (IKKβ) during EV71 HostfactorsmodulateEV71replication
infection[113]. EV71 genome contains a type I internal ribosome
IFNs induce the expression of ISGs to inhibit entry site (IRES) located at the 5(cid:4)UTR, which
virus replication in an autocrine and paracrine requiresanumberofhostIRES-specifictransacting
manner [114]. Accordingly, EV71 hinders the factors(ITAFs)toinitiateviralproteintranslation.
REVIEW
274 NatlSciRev,2015,Vol.2,No.3
It has been reported that four ITAFs, heteroge- miRNAs are also the targets for EV71 to escape
neous nuclear ribonucleoprotein K (hnRNP K), innateimmuneresponses[135,136].Forexample,
hnRNP A1, far-upstream element-binding protein EV71-induced miR-146a, a negative-feedback
(cid:4)
1 (FBP1), and FBP2, interact with the 5 UTR regulator in RLRs signaling, could inhibit IL-1R-
of EV71 to mediate virus replication [117–120]. associated kinase and TNF receptor-associated
hnRNP A1 and FBP1 play positive roles in ac- factor 6 activation, and further block IFNs
tivating EV71 IRES, whereas FBP2 is a negative production.
regulator.hnRNPKstimulatesEV71replicationby In summary, the mechanisms responsible for
interacting with the cloveleaf structure, stem-loop HFMD pathogenesis have not been fully under-
(cid:4)
II of the 5UTR, or with the stem-loop IV of the stood.StudiesonEV71showedthatmanyfactors,
IRES [117]. Recently, it has been reported that a includingreceptorbinding,viralfactors,innateim- D
o
serine/threonine kinase, Misshapen NIK-related muneevasion,andhostfactorsareinvolved.Abet- w
n
kinase,isinvolvedinIRES-mediatedtranslationof terunderstandingofthevirus–hostinteractionsas loa
d
EV71 by facilitating hnRNP A1 translocation to well as breakthrough in animal models is needed e
d
the cytoplasm [121]. Furthermore, EV71 impairs toprovideinsightsintothemechanismsunderlying fro
m
the processing of host pre-mRNA by cleaving the HFMDpathogenesis. h
cleavagestimulationfactor64Ksubunit(CstF64), ttp
s
which is advantageous for virus replication ://a
[122]. STRUCTURE-BASEDDESIGNOF ca
d
Upon RNA virus infection, intracellular host ANTIVIRALS em
membranesareremodeledtogenerateaviralRNA ic
replication center. Many host factors, including Given the devastating neurological effects that .oup
GTPaseADP-ribosylationfactor1(ARF1),Golgi- HFMD can have in young children, there is a .co
pressingneedtodevelopanti-EVagentstocombat m
specificBrefeldinAresistancefactor1(GBF1),acyl- /n
coenzyme A-binding protein domain 3 (ACBD3), HFMD. Although there are currently no available sr/a
and phosphatidylinositol 4-kinase IIIβ (PI4KB), antivirals to treat HFMD, virological studies have rtic
providedcriticalinsightsintoantiviraldevelopment. le
participateinthisprocesstoinfluencevirusreplica- -a
Theavailabletargetsforanti-EVcompounddesign b
tion[123–126].PI4KBcanberecruitedtotheGolgi s
membranesbyARF1orACBD3.Interestingly, T- can be categorized according to the target type, trac
00127-HEV1,asyntheticinhibitorofPI4KB,canbe rangingfromtheviruscapsidstructuralproteins,the t/2
usedtoinhibitEV71replication[124].Thissuggests viralencodednon-structuralproteins,andtheUTR /3/2
6
thatPI4KBmayplaypivotalrolesinEV71replica- of genomic viral RNA to host proteins implicated 8/2
invirusinfection.Eachtarget,inturn,corresponds 1
tion.Furtherinvestigationisneededtoelucidatethe 3
detailedmolecularmechanismsofthispathway.Ad- tocriticalstepsinviruslifecycle,includingvirusat- 036
ditionally,theendoplasmicreticulumproteinretic- tachment/entry/uncoating, virus protein synthesis 2 b
ulon3isanotherimportanthostfactorthatcouldaf- and maturation, RNA genome replication, im- y g
u
mune evasion, and virus assembly/morphogenesis e
fecttheEV71-encodedviralproteinssynthesisand s
viralRNAreplicationbyinteractingwiththe2Cpro- (Fig.4). t on
tein[127]. High-resolutionstructuralinformationisoneof 16
the essential resources for antiviral drug design, N
TheinteractionnetworkbetweenEV71andhost o
cells has been dissected by transcriptomic or pro- optimization, and validation. The structures of vi- vem
teomic approaches, which are helpful for elucidat- ral proteins encoded by EV71 have been stud- be
ing the pathogenesis of EV71. These results show ied extensively. Therefore, developing inhibitors r 2
0
against viral proteins can be an efficient path- 1
thatEV71infectioninducedchangesinmanyhost 8
way to develop HFMD drugs. We here summa-
processes,includinginproteintranslationandmod-
rize the progress in understanding the role of
ification, protein and ion transport, cell death, au-
certain viral components (mainly exemplified by
tophagy, and cell homeostasis [128–130]. These
EV71) in antiviral design in a structural biology
studies open the door for further highlighting the
view.
EV71andhostfactorinteractionandforexamina-
tionofhowhostfactorsaffecttheEV71lifecycle.
Interactions between EV71 and cellular miR-
Viralcapsidproteins
NAs have also been reported. EV71 infection
can induce alteration of miRNA expression and EVcapsidproteinsareinvolvedincellattachment,
form a unique miRNA profile [131,132]. Con- entry,anduncoatingprocesses,whichareamongthe
versely, miRNAs can regulate EV71 replication earliestconfirmedtargetsforantiviraldrugs.High-
by targeting EV71 genome or proteins [133,134]. resolutionstructuralanalysisofthematurevirions
REVIEW
Leietal. 275
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/n
s
r/a
rtic
le
-a
b
s
tra
c
t/2
/3
/2
6
8
/2
1
3
0
3
6
2
b
y
g
u
e
s
t o
n
1
6
N
Figure 4. Road mapfor targeting key events in EV71 life cycle.Key steps in EV71 life cycleprovide invaluable insights into drug design. These o
v
include virus attachment/entry/uncoating, virus protein synthesis and maturation, RNA genome replication, immune evasion, and virus assem- e
m
bly/morphogenesis. b
e
r 2
0
and natural empty particles indicate that the VP1 interaction with cellular receptor or subsequent 1
8
GH loop can act as an adaptor sensor for cellular uncoating. Crystal structures of EV71 complexed
receptor attachment and a critical generic mecha- by four different 3-(4-pyridyl)-2-imidazolidinone
nismforuncoating,providingnoveltargetsforan- derivatives revealed the structure–activity cor-
tiviral design [137]. High-resolution structures of relates. With the help of quantum mechanics-
EV71asemptycapsidandinhibitorboundvirional- enhanced ligand docking, the inhibitors were
lowed for the identification of a large cleft at each optimized,leadingtothesynthesisofacompound
icosahedralfaceofthevirusparticle,beneathwhere with an order of magnitude more potent antiviral
the hydrophobic floor serves as the docking site activity [140]. Rossmann and colleagues crys-
for drugs [138,139]. The binding of the drug at tallized EV71 virion complexed with inhibitor
the cleft ultimately rigidifies the virus capsid (dis- WIN-51711,oneofthe‘WIN’compoundsreferring
cussedindetailbelow),hencehinderingtheintrinsic toSterlingWinthrop,bywhomtheinhibitorswere
conformational dynamics that are essential for the developed. Rossmann and colleagues found that
REVIEW
276 NatlSciRev,2015,Vol.2,No.3
WIN 51711 replaced the natural pocket factor overby3Coritsprecursor3CD,thuscompletingvi-
andoccupiedthehydrophobicpocketontheVP1 ralproteinprocessing.Interestingly,2Aand3Cboth
protein. Furthermore, Win-51711 stabilized the also interfere with cellular functions. In addition
EV71virionandrestrictedthecapsiddynamicsthat toantagonizinghostinnateimmunityresponses,as
are required for genomic RNA release [139]. The discussed above, 2A can also shut down host pro-
determination of EV71 capsid dynamics provides teinsynthesisbytargetingeukaryoticinitiationfac-
the basis for capsid-binding inhibitor design and tor 4G1 (eIF4G1) to favor IRES-dependent viral
optimization. However, an obvious drawback of protein production [145,146]. The available crys-
a capsid-targeting agent is that most (if not all) talstructuresforEV71proteasesshowthat2Aand
of such inhibitors rapidly induce the generation 3C maintain the chymotrypsin-like fold and share
of drug-resistant progeny virus. This is due to the active site geometry for hydrolysis [147,148]. The D
o
‘prone-to-error’natureofRdRp.ThefactthattheEV structuresof2Aand3Carehighlyconservedindif- w
n
structuralproteinsareingenerallessconservedthan ferentEVserotypes,makingthemidealtargetsfor loa
d
the non-structural proteins also suggests the low wide spectrum inhibitors. Indeed, the human rhi- e
d
genetic barrier to the resistance of capsid-binding novirus(HRV)inhibitorRupintrivir,originallyde- fro
m
inhibitor. signedtodockthesubstrate-bindingpocketofHRV h
3C, was found to effectively inhibit EV71 replica- ttp
s
tionwithIC50atnanomolarlevel.Thestructuralba- ://a
2Chelicase sisforanti-EV71activitybyRupintrivirwasrevealed ca
d
bythecrystalstructureoftheEV71–3C-Rupintrivir em
2C helicase is one of the most conserved EV pro-
andCV-A16–3C-Rupintrivircomplexes[149,150]. ic
teins [141], but it remains one of the least char- .o
The structures show that Rupintrivir binds EV71 up
acterized to date. Collectively, EV 2C helicase is 3C similarly to its binding of HRV 3C (Fig. 5). .co
implicated to possess many key functions within m
rjuesptli∼ca3ti3o0n,aimmimnounaceidesv,aisniocnlu,dainngdvmirourspuhnocgoeanteinsigs,, Asinhlgathrueopu∼gth3h0et%hsuesbeHsqtuRraeVtnec-abeninidddeEinnVtgi7typ1,o3tchkCeetrpearsoriedteunaeesseasrblyuoinilndly-- /nsr/artic
covering nearly every step in the virus life cycle le
[113,127,142–144].TheN-terminalofEV712Cis variant.EV712Acrystalstructuresweredetermined -ab
asapoenzymeandanoctopeptidesubstratebound s
thoughttocontainanamphipathichelixthatcanan- form [151] revealed an open, shallow and flexible trac
chor the vesicle membrane, thereby recruiting the substrate-binding pocket, which is consistent with t/2
virus replication complex. EV71 2C also contains /3
thelowsubstratespecificityof2A.EV71proteinases /2
ttweromsienpuasr,awtheiRchNaAre-bbienldieivnegdmtoorteifcsoagtntihzeetNhe-acnlodveCr-- playpivotalrolesinreplicationandimmuneevasion 68/21
and they are one of the best studied EV proteins 3
leafstructureattheuntranslatedregionoftheviral 0
structurally.Thus,thecurrentknowledgeaboutEV 36
RNA genome. These potential functions allow 2C 2
proteolyticenzymecanreadilyfacilitaterationalde- b
helicasetobeapromisingtargetforthedesignofa y
signofnovelantiviralagents.Forinstance,arecent g
widespectruminhibitor.However,thelackofhigh- ue
studyshowedthatpeptidylaldehydeNK-1.8kwhich s
resolutionstructuralinformationforEV712Cleaves targets3CcaneffectivelysuppressEV71andEV68 t on
alargeknowledgegapforunderstandingitsfunction infections[152]. 16
onamolecularlevel,andhashinderedinhibitorde- N
o
sign. v
e
m
b
e
RNA-dependentRNApolymerase r 2
Virusproteases 01
RdRp is no doubt an important target for an- 8
Virus proteases are, in general, believed to be the tiviral development, as it is the catalytic unit for
most promising therapeutic targets, as evidenced virusRNAsynthesis.ThecrystalstructuresofEV71
by the successes of proteinase inhibitors in treat- RdRp complexed with nucleotide, nucleotide ana-
inghumanhepatitisCvirus(HCV)andhumanim- log, and the viral peptide (Vpg) have been re-
munodeficiencyvirusinfections.ThefactthatEVs solved[153,154],providinganessentialframework
encode two proteases (2A and 3C), which is rare forstructure-basedinhibitordesign.Structuralcom-
for its limited genomic size, indicates their impor- parison with the RdRps from foot and mouth dis-
tant role in virus infection. Despite their shared easevirusandCV-B3showssignificantsimilarityin
proteolyticactivity,thefunctionsof2Aand3Care overallfolding,nucleotiderecognition,orVpgbind-
not at all redundant. EV71 2A carries out the first ing,offeringevidencethatinhibitorstargetingother
cleavageofpolyproteinprecursoratVP1–2Ajunc- picornavirus RdRp may be also effective against
ture, whereas the subsequent cleavages are taken EV71.
REVIEW
Leietal. 277
curate structural information for rational drug de-
sign.Astherearenohomologspresentinmammals,
inhibitorstargetingenteroviralproteinscouldbea
prime choice concerning the safety issue. The un-
precedented success in treating HCV infection by
direct-actingantiviralsopensaneweraforcombat-
ingRNAvirusinfection.
VACCINEDEVELOPMENT
D
o
Protectiveneutralizingantibody(NAb)responseis w
n
one of the most critical host defense mechanisms loa
d
against viral infection [157]. Seroepidemiological e
d
dataindicatethatNAbsagainstbothEV71andCV- fro
A16areverylowinchildrenaged>6monthsto≤1 m h
yearsandgraduallyincreaseinindividualsbetween ttp
s
1-and4-yearoldage[158–160].Thesedatasuggest ://a
c
thatinfantsaged6–14monthsshouldreceiveprior- a
d
ityvaccinationagainstEV71. em
HFMD-associatedEVsinvadethehumanbody ic.o
throughmucosalsurfacesandinfectneuralsystem up
.c
afterviremiatocauseclinicalsymptoms.Avaccine o
m
against the main causing agents of HFMD should /n
s
be capable of inducing mucosal immune reaction r/a
Figure5.RupintrivirbindsEV713Cactivesiteinasimilarmodeasthebindingto to prevent virus infection at the first step and/or rtic
HRV3C.TheactivesiteofEV713Cisshownasasolvent-accessiblesurface.The le
colorofthesurfaceiscodedaccordingtoaminoacidconservationamongenterovirus of eliciting a system IgG reaction afterwards to -ab
block the viremia, a crucial step for neural system s
3bbCyouppnrrood.gRRreuespssiindivtuereivfsairidn(iynregeldltooawwre)haiinntevd,awtrhiahenictR,huaipnniddnitcirnaivctireresbatoshiuenngldeaatmostiHncooRnVasce3idrCvve(awdrihraeittsieiod)nuaeirses.inmEdVoi7cda1etl3eeCdd infeThctieopnr.otectingroleofsystemantibodiesinEV71 tract/2/3
intheactivesitebysuperimpositionoftheHRVandEV713Cstructures.Theinterac- vaccinedevelopmenthasbeenexemplifiedbyinac- /26
8
tionbetweenRupintrivirand3Cisprimarilymediatedbyinvariantresidues.Thebind- tivatedvaccines.Inthelasttwoyears,threeinacti- /2
1
ingpocketsfromS2’-S1’andS1-S4areindicated.Thedeviationsofconformationsof vatedEV71vaccineshavebeendevelopedforsys- 3
0
boundinhibitorsconformationaremostpronouncedattheterminiclosetotheS1’and tem immunization, and their safety and efficiency 36
2
S2’pockets,wheresignificantdifferenceswerealsoobservedbetweenEV713Cand have been verified in phase 3 clinic trials [161– b
y
HRV3C. 163]. These three vaccines share similar formula- g
u
e
tions and immunization protocols. Each is com- s
Few inhibitors target EV RdRp. One such in- prisedofformalin-inactivatedEV71virions(geno- t on
1
hibitor is ribavirin, a guanosine analog discovered typeC4)fromcellculturewithalumadjuvant.They 6
N
in 1972. The drug has been used in anti-HCV in- areadministeredtwotimeswithina28-dayinterval o
v
fectiontherapyfordecadesandshowsbroadspec- viaintramuscularinjection.Allofthethreephase3 em
trum against RNA viruses. Ribavirin was found to clinicaltrialsaremulticenter,randomized,double- be
be effective in reducing the fatality of EV71 infec- blind,placebo-controlledstudies,andincludemore r 2
0
1
tionbytargetingRdRp[155].TheefficacyofRib- than10000infantsandchildren(6–72monthsor 8
avirincouldbeattributedtoitsdirectinhibitionof 6–35months)wererecruitedforthetrial.Thesevac-
polymeraseactivity.Thesubsequentincorporation cines induce more than 99% seropositive rates af-
of Ribavirin into the virus genome induces muta- terboostingonceandprotect90–97%infantsfrom
genesis, which eventually leads to virus extinction. EV71-associatedHFMDin11–14monthsaftertwo
DTriP-22isanotherRdRpinhibitorthatisnotanu- intramusculardosesofEV71vaccines.Mostadverse
cleoside analog. DTriP-22 is able to inhibit EV71 eventsweremildandthetotaladverseeventsandse-
replicationbyreducingvirusRNAaccumulationin riousadverserateweresimilarbetweenthevaccina-
cells[156].DTriP-22-resistantanalysismappedto tionandtheplacebogroup.
aK163RmutationwithinRdRp,suggestingadirect AlthoughtheinactivatedEV71vaccinesaresafe
drug-RdRpcontact. and efficacious, there are some properties that re-
Insum,theviralproteinsencodedbyEV71have maintobeaddressed.First,asanti-EV71antibody
beenextensivelystudiedstructurally,providingac- titers decreased 6 months after vaccination in the