2014 The avian coronavirus spike protein

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VirusResearchxxx(2014)xxx–xxx

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Virus Research

journal homepage: www.elsevier.com/locate/virusres

The avian coronavirus spike protein

I.N.AmbepitiyaWickramasinghe1,S.J.vanBeurden1,E.A.W.S.Weerts,M.H.Verheije∗

DepartmentofPathobiology,FacultyofVeterinaryMedicine,UtrechtUniversity,Yalelaan1,3584CLUtrecht,TheNetherlands

a r t i c l e i n f o a b s t r a c t

Articlehistory: AviancoronavirusesofthegenusGammacoronavirusarerepresentedbyinfectiousbronchitisvirus(IBV),
Availab leonlinexxx theco ronavirusofc hic ken .IBVc ausesahighlycon tagi ousdisease affe ctingthe respiratory trac tand,

dependingonthestrain,othertissuesincludingthereproductiveandurogenitaltract.ThecontrolofIBV

Keywords: inthefieldishamperedbythemanydifferentstrainscirculatingworldwideandthelimitedprotection
Aviancoronavirus ac ross strai ns duetoser oty ped iversi ty.Thisd iversity isbelieved tobeduet oth eam inoaci dvariation
Gamm acoronavirus inthe S1dom ain of themajo rviralatta chm entprote in spike.In th el asty ea rs,m uche ffort hasbeen
InfectiousbronchitisvirusIBV
undertakentoaddresstheroleoftheaviancoronavirusspikeproteininthevariousstepsofthevirus’
Spike
livecycle.Variousmodelshavesuccessfullybeendevelopedtoelucidatethecontributionofthespikein
Binding
Tropism binding of the virus to cells, entry of cell culture cells and organ explants, and the in vivo tropism and
pathogenesis.Thisreviewwillgiveanoverviewoftheliteratureonaviancoronavirusspikeproteins

withparticularfocusonourrecentstudiesonbindingofrecombinantsolublespikeproteintochicken

tissues.Withthis,weaimtosummarizethecurrentunderstandingontheaviancoronavirusspike’s

contributiontohostandtissuepredilections,pathogenesis,aswellasitsroleintherapeuticandprotective

interventions.
©2014ElsevierB.V.Allrightsreserved.

1. Introduction Currently,itisworldwidepresentinbothindustrialandbackyard

chickens (reviewed by Cook et al., 2012; Jackwood, 2012; Sjaak

AviancoronavirusesofpoultrybelongtothegenusGammacoro- de Wit et al., 2011). IBV principally infects the epithelium of its

naviruswithintheorderNidovirales.Aviangammacoronaviruses hosts’upperairways,whichleadstorespiratorydistress,andpre-

cancausemajorhealthproblemswithsubsequenteconomiclosses disposes for secondary bacterial airway infections (Dwars et al.,

in several commercially kept bird species, predominantly chick- 2009;Matthijsetal.,2003).SeveralIBVstrainsadditionallyshowa

ens (Gallus gallus). The genus Gammacoronavirus comprises not subtype-dependenttropismforotherepithelia,includingtherenal

onlyvirusesofdomesticatedbirds,butalsotworecentlydiscov- tubuli,theoviductandpartsofthegastrointestinaltract(reviewed

eredcetaceancoronaviruses(Mihindukulasuriyaetal.,2008;Woo in Cook et al., 2012; Ignjatovic and Sapats, 2000; Raj and Jones,

etal.,2014).Inaddition,aviancoronavirusesbelongingtothegen- 1997).Thisresultsinvariablemorbidity,mortality,pathologyand

eraGammacoronavirusandDeltacoronavirushavebeendetectedin production losses in poultry. The great diversity of IBV strains

wild bird species (Chu et al., 2011; Woo et al., 2009, 2012), but worldwide makes it difficult to prevent infectious bronchitis in

detailsontheirpathogenesisandhostrangeareyetunknown.In chickens.ThepresenceofIBV-likeandotheraviancoronaviruses

thisreviewwewillfocusontheaviangammacoronavirusesofpoul- inotherbirdspecies(includingturkey,pheasant,quail,guineafowl,

try,inparticularontheroleofthespikeproteinintheoutcomeof partridge,peafowl,duck,gooseandpigeon)(Cavanagh,2005),com-

infection. plicatesthefieldsituationforaviancoronavirusesevenmore.

The avian infectious bronchitis virus (IBV) causes infectious IBVisanenvelopedviruswithapositivesensesingle-stranded
bronch itisin chickens.It istodate them ostim portant andbest- RNAge no m eof27.6kb (Mas tersa n dPerlm an,20 13).The5(cid:3)two-

studiedGammacoronavirusandisthereforeconsideredthegenus’ thirdoftheviralgenomecomprisesopenreadingframe(ORF)1ab,

prototype.IBVwasthefirstcoronavirusdescribed,andwasdiscov- whichencodesfor 15 nonstructuralreplicaseproteins(nsp2-16)
ered in the Un ites Sta tes in the 1930s (Schalk an d H awn , 1931). involv ed in RN A r epli cation and tra nscription . The 3(cid:3) one-third

of the viral genome codes for the structural proteins, which are

interspersedbytheaccessorygenes3a,3b,4b/intergenicregion,

∗ 5a,5b.Theseaccessorygenesaregroupspecificandhave,while
Correspondingauthor.Tel.:[phone].
beingdispensableforIBVreplicationinvitro(Casaisetal.,2005;
1 TEh-mesaeila audtdhroersss:c [email protected] t(oMt.hHis. Vweorrhke.ije). Hodgs onetal.,200 6),y etu nknownfun ct ionsi nvivo.T he stru ctural

http://dx.doi.org/10.1016/j.virusres.2014.10.009
0168-1702/©2014ElsevierB.V.Allrightsreserved.

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2 I.N.A.Wickramasingheetal./VirusResearchxxx(2014)xxx–xxx

proteinsofIBVincludethespikeproteinS,theenvelopproteinE,

themembraneproteinMandthenucleocapsidproteinN(Masters

andPerlman,2013).Aftergenomicreplication,theNproteinforms

together with the RNA genome the ribonucleocapsid, which is

encapsidatedbythestructuralproteinsE,MandStogeneratethe

virusparticle.

Thevirus’majoradhesionmoleculeisthespikeproteinS.The

characteristicsoftheSproteinaredescribedindetailinSection4.

Muchefforthasbeenundertakentoaddresstheroleofthespike

proteininvariousstepsofthevirus’lifecycle,andintheoutcomeof

infectioninvivowithrespecttotropismandpathogenesis.Various

modelshavesuccessfullybeendevelopedtostudydifferentstepsof

theIBVinfection.Thisreviewprovidesanoverviewoftheliterature

andrecentachievementsregardingthespikeproteinofaviancoro-

naviruses,tosummarizeourcurrentunderstandingonthespike’s

contributiontohostandtissuepredilections,pathogenesis,andits

roleintherapeuticandprotectiveinterventions.

2. Infectiousbronchitis

The disease described as ‘infectious bronchitis’ is a collection

ofsymptomscausedbyIBVsubtypes,whichcanbediscriminated

based on genotype, serotype and protectotype (Sjaak de Wit

et al., 2011). The classical subtype causing respiratory disease,

IBV Massachusetts 41 (M41), was isolated by Van Roekel in the

United States in 1941 (reviewed by Fabricant, 1998). Subtypes

other than M41 also cause respiratory disease, but with varying

severity. Respiratory disease is often clinically characterized by

dyspnea, coughing, rales and serous nasal discharge (Cavanagh

andGelb,2008).Itiscausedbyinfectionoftheciliatedepithelium

of the upper respiratory tract (mainly nasal cavity and trachea),

resultinginlossofciliaryactivity,degeneration,desquamationand

lossofthesecells.Inaddition,infectedtissuesshowshyperemia

andinflammation(Fig.1A),whichismainlycharacterizedbythe

presenceofheterophilicgranulocytesandlymphocytes(Fig.1B).

IBV can also spread to the lower respiratory tract and cause

aerosacculitis(Bezuidenhoutetal.,2011).Usually,theepithelium

is restored to normal within 2–3 weeks via a state of extensive

hyperplasia(Dwarsetal.,2009;Nakamuraetal.,1991;Purcelland

McFerran,1972).

Fromtherespiratorytract,thevirusspreadsthroughthehost
Fig.1. Macroscopic,histologicalandimmunohistochemicalanalysesofchickentra-
viaviremia(JonesandJordan,1972)totheepithelialcellsofthe
cheaofmock-infectedorIBVM41-infectedlayerchickens.Seven-day-oldSPFlayer
ren altubuli (Chen and Itakura, 1996; Con dro nandMar shall, 19 86; chick en swereoronasa lly inf ectedwithPB S(mo ck)orM4 1andsacrifice dat vari-

Purcell et al., 1976) and the ciliated epithelium of the oviduct oustimepointsafterinfection.(A)Longitudinallyopenedtracheaofmock(upper)
(Crinion e t al ., 1971 ; Jon es and Jord an, 1971). He re th e virus and IBV- M41 in fecte d (lower) chic ken at 7 dpi; t he M41 -infecte d t rachea shows
smallamountsofmucoidmaterialinthelumenandmarkedmultifocalhyperemia
causesrespectivelyrenalfailurewithurateobstructionduetotubu-
lar nec rosis with m onon uclear infla mma tion (Chen and Ita kura, iomf mthue nmohuicsotosac.h (eBm) iHstermysattaoixnyinlign( raingdh te)oosfians (eHct&ioEn) (olfetfht)e atrnadc haenatio-IfBaVm So2c kM-iAnbfe c4t8e.d4

1997;Jones,1974),andoviductalnecrosisandmalformationlead- (7dpi)orM41-infected(3dpiand7dpi)chicken.ThetracheaoftheM41-infected
ing to abnor mal eg g pr oduction and inab ility to lay (Chous alkar ch icken a t3dpishows an int acte pi thel iallining with minim al hyp eremia,while
bothcili ate d epit helialc ells andn on-ciliated mucu s-pro ducingep ithelialcells show
andRoberts,2007).Theseverityofthediseaseinvariousorgans
markedintracytoplasmicpresenceofS2antigen.At7dpi,thetracheahaslostnor-
dependsontheIBVsubtypeandultimatelydeterminesthemortal-
malarchitectureduetodesquamationoftheciliatedandnon-ciliatedepithelium
ityinchi cke ns. with replacemen tby ah yperplastic,mo re squ amousn on-c iliatedepith elium,infil-

MinorpathologicalchangesduetoIBVinfectioncanoccasion- trationbylargenumbersoflymphocytes,markedhyperemiaandinthesuperficial
allybese eninothero rgans.T hev iru sha sbeensh own toinfect layer p res ence o f necroti c c ells. The lume n conta ins desquam ate d e pith elial cells,
markednumbersofheterophilicgranulocytesandabundantmucoidmaterial.Both
glandular epithelial cells of the proventriculus (Yu et al., 2001),
aswellas cellsrese mblin gh isti ocytesandlymp hoi dc ells inand cthhea negpeitshoeblisaelr vliendinign tahnedm luomcke-nin sfheoctwe dcechllisc ckoennttariancihnega t.hSec aSl2e baanrtsigreenp.r eTsheenrte5 a0re(cid:2) nmo.

enterocytescoveringthececaltonsils(Owenetal.,1991).However,

thisdoesnotresultinsignificantclinicalgastrointestinaldisease.

IBV can also infect the Harderian gland (Toro et al., 1996, 1997;

vanGinkeletal.,2008),anorganinvolvedintheimmuneresponse. high sequence similarity to IBV or IBV vaccine strains (Liu et al.,

Finally, it has been reported that testicles can be infected, from 2005; Sun et al., 2007), others strains are much more divergent

whichIBVcanbevenereallytransmittedbythesemen(Gallardo andmayrepresentdifferentvirusspecies(reviewedinCavanagh,

etal.,2011). 2005). For example, turkey coronavirus TCoV is very divergent

Aviancoronaviruseshavebeendetectedinvariousotherpoul- in its spike gene, and causes in contrast to the respiratory dis-

try species. While some of these IBV- or IBV-like viruses display easeobservedforIBV,gastrointestinaldiseaseinturkeys(Meleagris

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I.N.A.Wickramasingheetal./VirusResearchxxx(2014)xxx–xxx 3

gallopavo). Swollen and edematous intestines with frothy con- Oviductorganexplants(Morketal.,2014)havebeendevelopedto

tent are the result of the observed severe enteritis (Cavanagh, studyIBVstrainswithtropismforreproductivetract.Finally,most

2001; Guy, 2000; Maurel et al., 2011). Quail coronavirus, which IBVfieldstrainsgrowonisolatedprimarychickenembryokidney

has a high spike sequence identity to TCoV, also causes enteri- (CEK)(Gillette,1973)cells(Fig.2B),althoughadaptationmaybe

tiswithsubsequentdiarrheaandreducedgrowthinyoungquails required.

(Coturmixcoturmix)(Torresetal.,2013).Inguineafowl(Numida TostudyIBVinalaboratorysetting,thecellcultureadaptedIBV

meleagris),guineafowlcoronavirusresultsinacuteenteritisand strainBeaudetteismostcommonlyused(BeaudetteandHudson,

pancreatitis(Liaisetal.,2014).OtherIBV(-like)coronavirusescause 1937). In contrast to most IBV field strains, Beaudette can infect

respiratory and kidney diseases in pheasants (Phasianus colchi- baby hamster kidney cells (BHK-21) and monkey kidney cells

cus)(Cavanagh,2005;Goughetal.,1996),andrespiratorydisease (Vero)(Fig.2B)(Maduetal.,2007;Otsukietal.,1979).Beaudette

and pancreatitis in pigeons (Columba livia; Qian et al., 2006). In wasobtainedbyserialpassagingofthevirulentMassachusettsM41

wildbirds,aviangammacoronavirusesaredetected,butwithout straininembryonatedchickeneggsandculturedcells(Beaudette

clinicalsignsintherespectivehosts(Cavanagh,2005;Chuetal., andHudson,1937).Asaconsequenceoftheadaptation,Beaudette

2011). lostitsabilitytoinfectchickensandisnotpathogenicforchickens

(Geilhausenetal.,1973).

3. Modelsystems The cell culture adapted IBV strain Beaudette has also been

the primary choice as backbone for the development of recom-

LaboratorystudiesonIBVarehamperedbytheinabilityofIBV binant viruses (Casais et al., 2001; Fang et al., 2007; Youn et al.,

fieldstrainstogrowincontinuouscelllines.Despitethis,severalin 2005). Only recently, a recombinant system based on the atten-

vitroandexvivomodels,inadditiontoinvivomodels,areavailable. uated H120 vaccine strain was generated (Zhou et al., 2013).

IBVcanbeculturedinintra-allantoicallyinoculatedembryonated Recombinantvirusesareexcellenttoolstodissectthecontribution

chickeneggs.Thevirusinfectsepithelialcellsofthechorioallantoic of each of the viral proteins to the various steps of the infec-

membrane(CAM)(Fig.2A)andthechickenembryo.Infectiousvirus tioncycle.Suchevaluationismuchmorecumbersomewithwild

particlesaccumulateintheallantoisfluid,fromwhichtheycanbe typeIBVstrains,wherefrequentlymultiplemutationsacrossthe

harvested.MorphologicalchangesoccurinboththeCAM(thicken- genomearepresentthatmayallcontributingtodiseaseinchick-

ing)andtheembryo(curling,stunting,growthretardation)(Loomis ens.

etal.,1950).Inaddition,exvivoorganexplantshavebeendevel- To fundamentally study the role of the spike protein in virus

oped,ofwhichtrachealorgancultures(TOCs)aremostcommonly binding,recombinantsolublespikeproteinshavebecomepracti-

used(Darbyshireetal.,1976).Asread-outparameterforinfectiona caltools.Proteinhistochemistryassaysinwhichbindingpatternsof

scoringsystemforciliostasishasbeendeveloped(Cooketal.,1999). differentIBVspikeproteinstovariouschickentissuescanbecom-

paredhavebeendevelopedbyus(Wickramasingheetal.,2011)and

others(Shahwanetal.,2013).Theseassaysalsoallowtheelucida-

tionofspecifichostattachmentfactorsinvolvedinIBVinfection,as

detailedinthefollowingsections.

4. Thespikeprotein

4.1. Characteristics

Thespikeproteinisthelargestofthecoronavirusstructuralpro-

teins,andconstitutesthecharacteristicclub-likeorpetal-shaped

16–21nmprotrusionsthatemergefromthevirionsurface,giving

itacorona-likeappearancewhenvisualizedbyelectronmicroscopy

(MastersandPerlman,2013).Thespikeproteinmonomerisatrans-

membraneglycoproteinwithamolecularmassofaround128kDa

before glycosylation (Masters and Perlman, 2013). A cleaved N-

terminal signal peptide (Binns et al., 1985) directs the S protein

towardtheendoplasmaticreticulum(ER),whereitobtainsexten-

siveterminalN-linkedglycosylation(Cavanagh,1983a,b).Around

thirtypotentialN-linkedglycosylationsiteshavebeenpredicted

fortheIBVspikeprotein(Binnsetal.,1985),increasingitsmass

to about 200kDa. After glycosylation in the ER, the monomers

oligomerizetoformdimersortrimers(Cavanagh,1983a,b;Delmas

andLaude,1990;LewickiandGallagher,2002).

TheSproteinofaviangammacoronavirusesiscleavedbyafurin-

likehostcellproteaseatthehighlybasicpentapeptidemotifRRFRR,

generatingthesubunitsS1andS2ofabout500and600aminoacids

Fig.2. Infectionofembryonatedeggs,cellcultureBHK21cells,andprimarychicken insize,respectively(Cavanaghetal.,1986a,b,c).AllreportedIBVS
emb ry onickidne y cells(CEK)wit hIBV Bea udettea nd-M4 1.(A) Ten -day-old embry- pr otein sequencesco ntainthisc le ava gerecogniti on site,andm ino r
onatedchic keneg gswe reino cula ted withBeaud ette andM 41 andchorioa llantoic
aminoacidvariationsarebelievednottocorrelatewithserotype,
membranes(CAM)werestainedwithanti-IBVS2MAb48.4.(B)BHK21andCEK
pathogenicityandtropism(Jackwoodetal.,2001).TheN-terminal
cellswereinoculatedwithIBVBeaudetteand-M41,fixedat8hpiandimmunofluo-
resce ncew asperform edu sing theanti-IB VN prote inMA b2 6 .1.I BVa ntigenswere S1subunitis part ofthela rgeectodo ma in andfo rms thebulbof

presentinBeaudette-inoculatedBHK21,CEKandCAM,whileforM41infectionwas theoligomericSprotein.TheC-terminalS2subunitcomprisesthe
BonHlKy2 o1ba snerd vCedE Kinc eClElsKw aenrde CtaAkMen. Sacta4le0 ×ba mrsa gfo nri fiCcA aMtiosn r. eprese nt 50 (cid:2)m ; picture s of oth er part of th e ectodom ain forming a n arr ow stalk , and the sh ort
transmembraneandendodomain.

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ThecoronavirusspikeproteinisaclassIviralfusionpeptide, Beaudette CK

in which the variable S1 domain is involved in host cell receptor 71 M41
bindingandtheconservedS2domainmediatesfusionofthevirion
andcell ular mem branes(B osc hetal., 2003;Ma stersa nd Per lman, 98 H120

2013).Allmappedreceptor-bindingdomains(RBD),includingthat Conn46

ofIBV(Promkuntodetal.,2014),arelocatedatvariouspositions
ArkDPI
withintheS1domain(MastersandPerlman,2013).TheS2mem-
branef usio nu nitoft heectodo mai ncontain stwoh ept ad repeat 94 Call99

regions(HR1andHR2),whichinteracttoformthecoiled-coilstruc- QX

ture of the stalk (de Groot et al., 1987), and a putative fusion
Pigeon
peptide.Afterendocytosis,conformationalchangesintheSprotein
aretrigg ered byexposure toacidicpHin endosom e s(C h uetal., Duck

2006), resulting in fusion of the viral envelope with the cellular Partridge
100
membrane.Incontrasttosomeothercoronaviruses,noendosomal
D274
proteaseshavebeenelucidatetocontributetotheinfectionofIBV

(see also Section 6.5). Although the S2 domain is not principally 4-91

involvedinbindingtoahostcellreceptor,theinterplaybetween B1648

S1andS2mightsynergisticallydeterminetheavidityandspeci-
Italy02
ficityofvirusattachment(deHaanetal.,2006;Promkuntodetal.,

2013). Turkey FR
Guineafowl

4.2. Sequencediversity
100 Quail
Incontrasttovirusspeciesbelongingtothealpha-andbetacoro- 90 Turkey US

naviruses,whichoccurasonlyoneortwodifferentserotypes,there

aremanydifferentserotypesofthechickenGammacoronavirusIBV
0,1
(Jackwood,2012;SjaakdeWitetal.,2011).Asthemainantigenic

viralproteincontainingepitopesforneutralization(Cavanaghetal.,
1986 a,b,c;Ka ntetal.,19 92;Koch et al.,1990;Moc kettetal.,1 98 4; tFhige. s3p.i kPehpyrlootgeeinneftoicr saenleaclytesids aovfi atnheg aammminaoc oarcoinda sveirquuseensc.eSse qoufe tnhcee sSw1 esruebaulnigint eodf

Niestersetal.,1987),thehighsequencediversityoftheS1domain usingMUSCLEwithdefaultsettings.Theunrootedmaximumlikelihoodtreewas
accounts fo rth issero typi calv ariation.W hileami no acid se quence constr ucted usi ng M EGA 6.06 with the best -fit substi tution mod el (LG+G), w ith c om-
identityf ort hec onservedS2 domainis usual ly≥90% bet weendif- plete deletion of gaps. Bootstrap values (500 replicates) are indicated at the nodes
when>70.
ferentserotypes,variationintheS1domainrangesfrom2–3%up

to50%,withanaverageof20–25%(BrittonandCavanagh,2007).
Ap hylo gene tic treeofS1 p roteinso fvarious IBV reference strains probably includes selective pressure, high virus titers and high
is showninFig .3,s ho win gtherel ati onshipa mo ngstthes elected viral sub populatio n diversit y (Jackwo od e t al., 2 012). Intra host
av iancor on aviru s speciesw ith regardtothe spike. selec tion of specific IBV sub populations m ay be the result of
Th ereisacorre lationb etwe enthe am ino acidsequenceiden- the chick en immune res ponse, affinity f or ho st rece ptors a nd
tityandt he l evelofcross -protecti on: IBVstra insw ithinthe same mic roenviro nmental differences betwee n o rgan systems ( Toro
sero type us ually sh are more than 95 % am ino ac id iden tity in S1 etal.,2012a,b).Theem ergenceof newIBVv ariants issubseq uently
(Cavanag h,2001) ,wher easIBV stra inso fothers erot ypesshar el ess fac ilit atedbyth ehi ghbasicrep ro duct iven umber, es timatedtobe
than 85% a mino a cid iden tity (Cavan ag h, 200 5). Howe ver, so me 19.95(deW it eta l.,19 98).T hewide-spre aduseof live-attenu at ed
highl ysim ilarstr ains showon lylimitedcr oss-pro tection,w hilsta vaccin est rain sa nd thesub seq uentselectiv epr ess ureinducedby
high le vel of c ross-pr otecti on m ay exis t for strains with a muc h neutrali zing an tibo dies against the spike m ay force t he adap ta-
lowe rhom olo gy(reviewedin Sjaak deW itet al.,201 1). tion of the v irus to esc ape imm uni ty, and hen ce res ult in faster
Usi ng monoc lonal anti bo dies, five co nfo rm ation-dependent evol uti onar yrate s(a sdiscu ssedinJack woo detal., 2012) .
neutraliz ingantigenic siteswerem appe donS1,aswellasanother Nexttom utatio ns, geneticre co mbination m ay leadtothecre-
immunodom inantreg ionin theN -termina lre gio no fS2( Ko chetal., ationan de mergenceo fnewge neticvariantst hatar ever yd iffe rent
1990;Kustersetal. ,1989; Le nst raetal.,198 9).The fiv en eutral iz ing from thep arentalstr ain s(M astersa ndPerlm an,2 01 3).Th eoccur-
antige nicsites o nS 1co-lo catewi th int hreeh yper vari ableregions rence ofr ecombin ationh asbeenre port edforma nycoro nav iruses,
(HVRs)(C avan agh et al.,1988, 1992; Moore etal.,1997;N iesters withs o- calledrecombin atio nhot spotsrep res enting regionsinthe
etal.,19 87),sugge stin gth eHVR stobe involv ed ina ntigen icityand geno mewithh igherrecombin atio nbre akpointincide nce.Su ch hot
he nce seroty picalvaria tion .The nu cle otideseq ue nceof(part of) spotsha vebe enma ppedforIBVto nsp2,nsp3 ,nsp16an dimm e-
theS1 subunitoft hespikeha stra ditionallyb eenusedf or genot yp- diatel y ups tream of the spik e g ene (Tho r et a l., 201 1). T he last
ing (OI E,2013) .F ord iagno stic purposes,gen otyp ingo fIB Visolates mentio nedhotsp ot isof specia linter est,as th eS protein cont ains
isu sually limite dto thehyperv ariableam inoterminu sr egio nofS1. viralneutra lizin gep ito pe sandd etermine st ropi sm .Hence ,recom-
As aresul tofthe gr eat geneticvariab ilityof IBVvarian ts,ad efi ni- binat ionoftheSg enemay resu ltintheem ergence ofnew strains,
tiv e nomen cl atur e and genetic classificat ion sy stem is, h o wever, newsero ty pes ,o reve nne wviru se sin fectingothe rh osts .Itwas
still lacking. hypo thesizedth at aone- time double cross-over event betwe en IBV
T he high sequence diversity of the IBV S1 domain and the andayetunk now n other(av ian)co ronavirusu poni nfection ofa
hyperv ariabl e amino terminus in par ticul ar ( Cavanag h et al., sing le hos tledtothe emer genceo fTCoV(Jackw ood etal.,201 0).

1998; Lee and Jackwood, 2001), is thought to be the result of

accumulation of mutations and selection. The average rate of 4.3. Spikesofotheraviancoronaviruses

synonymousmutationsinthegenomesofcoronavirusesisabout
1.2×10−3su bstitutions /sit e/ye ar(Hanad ae tal.,2004).Th er eason Although IBV has been described primarily as a chicken

forthehighsequencediversityoftheIBVspikeisunknown,but pathogen,IBVorIBV-likegammacoronaviruseshavebeenfoundin

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Fig.4. BindingofS1proteinsofdifferentIBVstrainstotracheaandotherclinicallyrelevanttissues.RecombinantS1proteinsofIBVM41,H120,Beaudette,B1648andQX

wereproducedandattachmentprofileswerecomparedbyperformingspikehistochemistryaspreviouslydescribed(Wickramasingheetal.,2011)ontrachea(A)andother

chickentissues(B).BindingoftheS1ofthevirulentMassachusettsM41straincorrespondedwiththecellsreportedtobesensitiveforinfectionwithM41.Specifically,M41

S1hadagreaterbindingaviditythanthatoftheattenuatedvaccinestrainH120ontracheaandlung(Wickramasingheetal.,2011).ForBeaudetteS1noobviousbinding

couldbeobservedonchickentrachea(Wickramasingheetal.,2011),butalsonotonCAM(Promkuntodetal.,2013).TheS1ofthenephropathogenicB1648didnotshow

anyappreciablestainingofthetracheaandthekidney,whilefortheS1ofQX,astrainwithreportedreproductivetracttropism,onlymildpatchystainingtotheoviductwas
obse rved.Scale barsrepr es ent 50(cid:2)m; (C)s che maticre presen tat ion of the abi li tyofre com binanttri mericspikep rotein stobind toa selec tedset oftissue sa ndt herepo rted

abilityofthecorrespondingIBVstrainstoinfectthesetissues;na:datanotavailablefromliterature;nd:notdone.

peafowl,partridge,blue-wingedteal,pigeon,mallardduck,gray- cellsandtissues,andtheroleofthespikeindefiningtropismand

lag goose (Cavanagh, 2005), pheasant (Cavanagh et al., 2002), pathogenicity.

guineafowl (Cavanagh, 2005; Liais et al., 2014) and various wild

birdspecies(Chuetal.,2011).Mostoftheseviruseshavenotbeen 5.1. Intissuebinding
isola ted,but were d ete ctedan dpar tly chara cterized bym ole cular

methodsonly. The effect of sequence diversity in S1 proteins between IBV
Poultr y other than chicken from which avian gammacoron- strains onbin din gwascle arlydemo ns trat edbyapp lyingrec om-
avirusesha vebee niso latedinc ludet urkey,q uaila ndguineafowl binant S1 proteins to c hicken tissues using s pik e histoch emistry
(Cavanag h,20 05;Ci rcellaet al.,2007 ;Liaiset al.,20 14; Torrese tal., (Wickr am asinghe et al., 2011 ) (Fig. 4). Th e bin ding avidity of
2013).TCoV isth oughtto o rig inatef rom a rec ombin ationo fI BV recombinant trim eri c S1 prote ins of IBV Ma ssachuse tts stra ins
witha genef ro munkno wn origin(Ja ckwo od etal.,2010).IB V and M41, H120 a nd Beau dett e correla ted wit h the reported patho-
TCoV f ullge nome sareabou t86% similar,wh ile th esimil arity for genic ity of thos e strains in vivo (Wic krama sing he et al., 2011).
theS gene islesstha n3 6%.Ext ensi vegenet icvaria tion intheSge ne Strikingl y, trimer ic solub le S1 p roteins of other st rai ns, includ-
sug ge stst ha tNo rthA mer icanandE uropean TCoVss ub seq u ently ingthenep hropatho genicB 164 8andthe QX causi ngrepro ductive
evolvedd iffer ently( Fig.3;Mau rele tal.,2011) ,result inginthecre- trac t d isorders, hardly sh owed any app rec iable bin ding to tra-
ation of different s eroty pe s (Jackw o od et al., 2010). Th e gen ome chea (Fig.4A)an dother clinicall yrele vantorgans ,likekidn ey and
of gu ine a fowl co ronavirus aligns to th e TCo V geno me, suggest- ovidu ct(F ig.4 B).O nlyfor IBVQXli mitedsta iningof theo viduct was
in gacom mon ancestor,but acurr en tsep arate evolution arypath observe d.An ove rview of the ob servedb indingo fv ari oustrim eric
(Lia is et al., 20 14). The S1 o f quail co ronavirus has ∼80% a mino spikeprot ein stotissue sa ndt heprevio uslyrep or tedinfec tivityof
acidi den tity withc ertai nT Co Vstra ins,andonly ∼17 %aaid entity thatp articular tis suebyt hec orre spondingIB Vstrain isdepicted in
with IBV.Ho weve r,asthe resto fthege nom eof quailc oro navirus Fig.4 C.
arey etto bedeterm in ed, itisn ot kno wnwhe th erqua ilcoronavi- W henexpressedasdimersfusedtothehumanFctail,M41S1
rus evo lve dw ithguineafo w la ndt urkeyc oronaviru sors eparately. showedm uchlessav idi tythan trimer ic M41 S1forc hic ken trach ea,
S1s equence sfro mcoronavi ruse sdetec tedinsome oth erpoultry whileB1 648S 1di mersg ained ,toalim ited ex ten d,thea bilityto
spe cies, inclu ding t hat of pigeon , partridg e a nd du ck, ar e much bind( Fig.5). The seresul tsarein lin e withth eprevio usly observ ed
less dive rgent from the S 1 of IBV reference stra ins (Fi g. 3) . It is, bind ingp atte rnsof dimeri cS1 of B16 48an dQX tooviduct explants
how ever,unkn ownw het her IB Vha sabroader host-ra nget han cu r- (Morke tal.,201 4) andoth er IB Vperm issi vec el ls,inclu dingtra-
rentlytho ught,orth atthese var ious b irdspec ieswerein fecte dby cheale pi thel ialcel lsan dprim ary chickenkid neyc ells(Shah wan
anum berofyet un cha racter izedIBV -like viruses . etal., 2013).Sim ilarly ,dim ericBea udetteS1 bound toch ickencells

(Hesseetal.,2012),whiletrimericBeaudetteS1wasnotsufficient

forbindingtotrachea(Fig.4A;Wickramasingheetal.,2011)and

5. Theroleofthespikeprotein CAM(Fig.4B;Promkuntodetal.,2013).Differencesinamountof

staining between laboratories might be explained by the use of

As argued above, the spike displays high sequence diversity 20-foldhigherproteinconcentrationsbyShahwanandcoworkers

amongstallcirculatingIBVstrains.Itisthereforebelievedtoplaya (Shahwanetal.,2013)comparedtothatusedbyWickramasinghe

crucialroleintheoutcomeoftheinfection.Belowwewillprovide etal.Nevertheless,itisclearthatthemultimerizationstateofthe

anoverviewoftheliteratureonaviancoronavirusspikebindingto spikeaffectsthebindingcharacteristics.

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Fig.5. BindingcharacteristicsofrecombinanttrimericanddimericIBVS1proteins.

RecombinantS1proteinsproducedinframewiththeGCN4trimerizationmotifand

theStrep-tag(‘trimers’)orinfusionwiththehumanFctail(‘dimers’)wereapplied

tochickentrachea.UsingsimilarproteinamountsfordimericandtrimericS1(ata

concentrationof0.1mg/ml),trimericM41.S1boundwithhigheraviditytotrachea

comparedtodimericM41.S1.Incontrast,dimericB1648.S1showedlimitedbinding

totrachea,whilefortrimericB1648.S1nobindingatallcouldbedetected.Scalebars
rep resent5 0(cid:2)m .

Fig.6. IBVspikedomainsinvolvedinbinding.Spikehistochemistrywasperformed

usingrecombinantsolublespikedomains.(A)TheN-terminalS1domain(M41.NTD,
With similar spike histochemistry assays, the contribution of
thespike domai nstob indingwaseluc idated. The N-terminal2 53 pcorimsipnrgisaiang2 7a3a –1593–227o2f tohfe thspe iskpei)koef) Man4d1 twhee rCe-taeprpmliiendalt oS1c hdiocmkeanint r(aMc4h1ea.C.TTDh,e cNomTD-

amino acids of the S1 of the IBV Massachusetts strain M41, but wasbothsufficientandrequiredforattachmenttochickentissues,andthuscon-
not the C-te rm inal dom a in (F ig. 6 A) had bound to cells in an (cid:3)- taine dth ereceptor -bin dingdom ain (Promkunto d etal.,20 14);(B )rec ombi nant
S1and ecto domainsofBeaud etteand M41werepr odu ced assolu ble recombinant
2,3-sialicaciddependentmanner(Promkuntodetal.,2014).Using
proteinsandappliedtoBHK21cellsandchickentrachea,respectively.Bindingof
thedifferencesinbindingbetweenM41andBeaudetteS1proteins
ami noacids38 ,43 ,63,and 69,partl yove rlap pingwithH V R1,were eBxetaeundseiottne osfpSik1ew tioth ctehlle cSu2lteucrteosd owmasa ionnfloyr oMb4se1rivnecdre faosre tdhteh eecbtionddoinmgaainv,i dwithyilteo tthhee

identifiedascriticalresiduesforbindingofM41(Promkuntodetal., trachea(similarlyasobservedbeforefortheCAM;Promkuntodetal.,2013).Scale
2014).IBV M 41isthe onlyGa mm acorona vir usfor whichtheRB Dh as bars rep resent 50 (cid:2)m .

beenidentified.Likelyimportantbindingpropertiesalsoresidein

thisdomainofotheraviangammacoronaviruses.Thecontribution There is contradictory data published on the cause of the

ofdomainsoutsidetheS1becameclearwhenstudyingBeaudette extendedhostrangeofBeaudette.Majorputativefactorsthatare

spikebinding(Promkuntodetal.,2013).WhileBeaudetteS1was studiedincludeadditionalcleavageoftheSproteinbyhostpro-

notsufficientforbindingtochickentrachea(Fig.6A),CAM(Fig.4B) teases and extended binding to host attachment factors, which

and BHK21 cells (Fig. 6B), extension of this protein with the S2 arebothdiscussedinSection6ofthisreview).Inaddition,ithas

part of the spike ectodomain resulted in binding to BHK21 cells beenshownthatparticularmutationsintheSproteinthatdrive

(Fig.6B)andCAM(Promkuntodetal.,2013).Interestingly,exten- thecell–cellfusionactivitydeterminetheinfectivityofBeaudette

sionoftheM41spike,nowcomprisingthecompleteectodomain, (Yamadaetal.,2009).Itmightwellbethatnotonlyattachmentto

resultedinanincreasedaffinityandextendedbindingprofiletothe additionalhostfactors,butalsofusionandperhapsotherdown-

chickentrachea(Fig.6B),andthusS1andS2mightworktogether streameventscontributetothecellculturetropismofBeaudette.

todeterminetheavidityandspecificityofvirusattachment.

5.3. Ininvivotropismandpathogenesis

5.2. Incellculturetropism

WhiletheS1domainisimportantinbindingandtheS2domain

The IBV spike glycoprotein is a determinant of cell tropism, contributestodeterminingtheinvitrotropismofIBV,theroleof

and the extended host cell tropism of Beaudette (see Section 3 theSproteininvivoislessclear.Exchangeofthespikeproteinof

of this review) is merely determined by this protein. By replac- BeaudettebythatofvirulentM41and4/91IBVstrainsintheviral

ing the ectodomain of the spike of Beaudette by that of M41 in Beaudette genome resulted in apathogenic recombinant viruses

the Beaudette viral background using recombinant IBVs (Casais BeauR-4/91(S)(Armestoetal.,2011)andBeauR-M41(S)(Hodgson

etal.,2003),itwasobservedthattheBeauR-M41(S)acquiredthe et al., 2004); the latter only showing poor replication in trachea

celltropismofIBVM41.WhileM41,recombinantBeaudetteand and nose compared to M41 (Hodgson et al., 2004). In addition,

BeauR-M41(S)hadtheabilitytoproduceprogenyvirusonprimary replacement of only the S1 domain of Beaudette by that of the

chickenkidneycells,theVero,BHK-21andCEFcellsonlysupported vaccinestrainH120renderednonpathogenicvirusthatcouldbe

infectionandreplicationofBeaudette(Casaisetal.,2003).Arecom- isolatedfromtrachealswabstillday6(Weietal.,2014),indicating

binantBeaudetteinwhichtheS1geneofBeaudettewasreplaced thattherecombinantvirushadtheabilitytoinfectandreplicatein

by that of H120 was recently generated (rBeau-H120(S1e)) and therespiratorytract.Toconcludetowhatextendthespikeprotein

retaineditsabilitytogrowonVerocells(Weietal.,2014),indicat- determinesinvivotropismclinicalstudiescomparingtheorganand

ingthattheprerequisitesforinfectionofcellculturecellsresides cellpreferencesoftherecombinantandparentvirusesareneeded.

inS2sequenceofBeaudette. The previously mentioned studies nevertheless suggest that the

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spike properties do not fully determine pathogenicity of IBV in In particular, glycan array studies revealed that the S1 protein

chickens. of the IBV Massachusetts M41 strain specifically bound the
Further elucidation of the contribution of the spike to tissue di- sialo date d glycan Neu 5Ac(cid:3) 2,3Gal(cid:4) 1,3(Neu5Ac(cid:3) 2,3Gal 1,4)-

tropismandpathogenesisinvivocamefromstudiesreportingintra- GlcNAc(Wickramasingheetal.,2011).AbdElRahmanetal.(2009)

host variation. Viral subpopulations with amino acid differences suggestedthattheobservedvariationintissuetropismbetween

in S1 distinct from the predominant vaccine populations were differentIBVstrainsmightbeexplainedbyadifferenceinprefer-

selectedinthetrachea(Gallardoetal.,2010;McKinleyetal.,2008; enceforspecificsialicacids.Inthisrespect,noparticularsialicacid

vanSantenandToro,2008),tearsandHarderiangland(vanSanten couldbeidentifiedinglycanarraysapplyingS1proteinsofother

and Toro, 2008), and reproductive tract (Gallardo et al., 2010). IBV strains, including H120, B1648 and QX (data not published).

These subpopulations were suggested to have different pheno- This suggests that either these proteins bind with much lower

typesacrossorgans(Gallardoetal.,2010;Ndegwaetal.,2014),and affinitiestothesamesialicacid,orthattheyrecognizeyetother

theirselectionmightdependontheIBVspecificimmuneresponses unidentifiedhostfactor,includingspecificsialicacidswhichmight

(Ndegwaetal.,2014;Toroetal.,2012a,b). benotpresentonthisarray.WhiletheresultsofMorketal.(2014)

ThenonstructuralreplicaseproteinsencodedbyORF1abclearly suggest that binding of recombinant dimeric S1 proteins of IBV

contributetothefitnessandpathogenicityofIBVinvivo.Arecombi- strainswithdifferentpathogenicitytosialicacidsiscomparable,

nantIBVcomposedofthestructuralregionofthevirulentM41and ourstudiesusingtrimericS1proteinsindicatethattheremightnot

thereplicasegenesofthenonvirulentBeaudette(Armestoetal., only be differences in binding characteristics of different spikes

2009) did not show any clinical symptoms after inoculation in (Fig.4),butalsodifferencesintheattachmentprofilesofthespike

chickens,andtracheasofallbirdshad100%ciliaryactivity,incon- protein to tissues from different animals. For example, binding

trasttoM41.NovirusorviralRNAcouldbedetectedinthetrachea of the same trimeric M41 S1 protein to oviduct of broiler and

ofbirdsinfectedwiththerecombinantBeauR-Rep-M41-Struct-2. layerchickens(Fig.7B)showedremarkabledifferencesinbinding

Itisofinteresttoelucidatewhetherthisviruscanreplicateatthe patterns,suggestingdifferencesinattachmentfactorexpression.

inoculationsite,andtodeterminewhetherthelackofpathogenicity Severalothercoronavirusesusehostsurfaceglycanscontain-

wasduetoreducedfitness.Furtherevidenceontheroleoftherepli- ing sialic acids as attachment factor (Schwegmann-Wessels and

casegenesinpathogenicitycamefromastudyinwhichthegenome Herrler,2006).Ithasbeenshownthatsialicacidbindingcanaffect

sequencesofvirulentandavirulentArkDPIIBVviruseswerecom- thetropismorthepathogenicityofthevirus.Forthemurinebeta-

pared(Ammayappanetal.,2009).Nexttothespike,mostamino coronavirusMHVextendedattachmenttoO-acetylatedsialicacids

acidsubstitutionswerelocatedinORF1a.Theproteinsencodedby hasbeenshowntoaffectcelltropism,resultinginincreasedneu-

thisregioninothercoronavirusesareknowntohaveanimmune rovirulence(Kazietal.,2005).Thealphacoronavirusesporcineres-

regulatoryroleanddeterminetheviralreplicationrate.Thus,they piratorycoronavirusandrelatedtransmissiblegastroenteritisvirus

mightbeinvolvedincontrollingthehostresponsetoinfection,or bothprincipallyuseacellularproteinreceptor,buttheextended

thefitnessofthevirus.Thereplicaseregionis,however,notthe abilityofthelattertobindtosialicacidislinkedtoitsenterotropism

soledeterminantofIBVpathogenicityasreplicasegenesequences (Krempletal.,1997;Schultzeetal.,1996).Interestingly,IBVdoes

ofIBVsrecoveredfrominfectedchickenswereidenticaltothose not express a receptor-destroying enzyme (Winter et al., 2006),

of vaccine viruses, with a lack of correlation with the pathotype whichisincontrasttoavianinfluenzavirusandcertainbetacoron-

(MondalandCardona,2004). aviruses(Schwegmann-WesselsandHerrler,2006).Tocompensate

fortheobservedlowerbindingaffinityoftheIBVspikecompared

totheHAattachmentproteinofavianinfluenza(Wickramasinghe
6. Hostdeterminants
etal.,2011)otherhostattachmentfactorsmightbeinvolved.

Thebindingofthecoronavirusspikeproteintoahostfactor,and
6.2. Proteinreceptors
thesubsequentfusionofthevirusandhostcellularmembranes,

arethefirststepsinvirus’lifecycle.Themainhostfactorthatis
Sofar,fornoneofthegammacoronavirusesafunctionalprotein
involvedinIBVattachmentisalpha2,3-linkedsialicacid.Inaddi-
receptor has been identified. For many alpha and betacoron-
tion,specificlectins,heparansulfateandcellularfurinhavebeen
aviruses, specific protein receptors have been identified that
showntoplayaroleinBeaudetteinfectioninvitro.Whereknowl-
restrict the host and tissue infection. Several of the alphacoro-
edgeonhostfactorsinvolvedinIBVinfectionislimited,suchas
naviruses (including porcine, feline, canine and certain human
withregardtothepossibleexistenceofaproteinreceptor,available
coronaviruses) utilize aminopeptidase N (APN) as receptor pro-
dataforothercoronavirusesisshortlyreviewed.Theinteraction
tein,whiletheproteinreceptorusageofbetacoronavirusesismore
between the spike protein and its specific attachment factors is
variable (Masters and Perlman, 2013). Murine coronavirus uses
consideredtobemainlyresponsiblefortherestrictedhostspecies
CEACAM1 as receptor (Masters and Perlman, 2013), the human
rangeandtissuetropismofcoronaviruses.Hence,thedistribution
Alphacoronavirus NL-63 and the betacoronavirus SARS-CoV both
ofthereceptorsiscriticalfortheoutcomeofinfectioninvivo.
useACE2(MastersandPerlman,2013),whilethebetacoronavirus

MERS-CoVusesDPP4(Rajetal.,2013).Migueletal.(2002)sug-

6.1. Sialicacids gested a role for APN during IBV entry, but Chu et al. (2007)

concludedthatfelineAPNwasnotafunctionalreceptorforIBV.

Alpha2,3-linkedsialicacidshavebeenidentifiedasareceptor Whetherthereactuallyisacellsurfaceproteinreceptortodeter-

determinant for IBV in both cells and TOCs (Abd El Rahman minethetropismofIBVremainstobeelucidated.

et al., 2009; Winter et al., 2006, 2008). Binding of alpha2,3-

linked sialic acids to specific lectins corresponded with the 6.3. Lectins

susceptibilityofthesecellstoIBVinfection,anddesialylationby

neuraminidase hampered infection. Our lab (Wickramasinghe OverexpressionofthehumanC-typelectinsDC-SIGNorL-SIGN

et al., 2011) and others (Mork et al., 2014; Shahwan et al., in normally nonpermissive cell lines enhanced infection of IBV

2013) demonstrated that binding of recombinant S1 proteins in a sialic acid independent manner (Zhang et al., 2012). Similar

to chicken tissues ex vivo (including trachea, lung, intestine and secondorco-receptors,besidesthementionedproteinreceptors,

kidney) also depended on alpha2,3-linked sialic acids (Fig. 7A). havebeenidentifiedforthehumancoronavirusesSARS-CoV(Jeffers

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Fig.7. BindingofM41.S1issialicaciddependentanddiffersbetweentheoviductofbroilerandlayer.SpikehistochemistryusingrecombinantM41.S1proteinswasperformed

asdescribedbefore(Wickramasingheetal.,2011)onsectionsof(A)tracheaofbroilerand(B)oviductsoflayerandbroiler.BindingofM41.S1couldbeinhibitedbypretreating
th etracheaw ithne uraminidasefrom Vi brio choler ae (VCNA)a nd by pre-inc ub atingth esp ike withthe N eu5A c(cid:3)2, 3specifi cglycan S iaLec(da tanot pu blished). Wh ileM41.S1
has highbin ding avidityforovid ucto fthela yer,bind ingtob roile ro viductalmostl acki ng.Sc aleb arsr epresent50 (cid:2)m.

etal.,2004;Marzietal.,2004;Yangetal.,2004),229E(Jeffersetal., (Cavanagh,1983a,b),andwassuggestednottocorrelatewithpath-

2006)andNL63(Hofmannetal.,2006),andforthefelinecoron- ogenicityoftheIBVstrain(Jackwoodetal.,2001).Anadditional

avirusesFIPV(ReganandWhittaker,2008)andFeCoV(Reganetal., furin-cleavagesiteintheS2subunitoftheBeaudettewassuggested

2010).However,asDC-SIGNandL-SIGNaremainlyexpressedon tobeinvolvedinthefurin-dependententryandsyncytiumforma-

nonepithelialcells,likedendriticcellsandendothelialcellsofliver tioninvitro(YamadaandLiu,2009).Thiscorrespondedwiththe

and lymph nodes, respectively, the role for chicken homologues observationthataproductivenessofIBVBeaudetteinfectioncor-

of human DC-SIGN/L-SIGN in IBV pathogenesis in vivo is hence related with cellular furin expression (Tay et al., 2012). Yamada

speculative. andLiu(2009)hypothesizedanovelXXXR/Scleavagesiteinthe

IBVspike,correspondingtothepositionoftheadditionalS2cleav-

6.4. Heparinsulfate agesiteinBeaudette,forcleavagebyotheryetunidentifiedhost
prot ease .S uchahost prot easemig htc ontrib ute tothetropis mof

NexttothepotentialcontributionoftheS2domain(Section5.1), IBVstrain,asithasrecentlybeenobservedthatBeaudetteentry,
the exte nd ed host trop ism of the ce ll cul tur e adapte d IBV st rain whi le bein g fur in-d ependen t on Vero cells , dep ended on a yet
Bea udettemig htas wellbe ex plai ned bybindi ngtoan add itional unide ntified proteaseinchick enk idney cells (C.Winter ,pe rso nal
cellsurfac erecep to r,nam el yheparan su lfate(Ma du et al.,2007). communicat ion).

Thisisawell-knownreceptorformanydifferentviruses(Liuand

Thorp, 2002), including a murine coronavirus MHV mutant with 7. Vaccinedevelopmentandprotection

extendedhostrange(deHaanetal.,2005).Uponpassageincell

culture,bothIBVBeaudette(Maduetal.,2007)andMHV(deHaan Thespikeproteininducesbothhumoralandcellularimmune

etal.,2005)adaptedbytheacquisitionofheparansulfate-binding responses in chickens infected with IBV (Cavanagh, 2007). The

sites,whichenabledtheuseofheparinsulfateasalternativeattach- presence of antibodies does not necessarily correlate with pro-

ment/entryreceptor.Theputativeheparinsulfate-bindingsiteof tection against IBV (Cavanagh et al., 1986a,b,c; Collisson et al.,

Beaudette overlaps with a second furin cleavage site in the S2 2000; Ignjatovic and Galli, 1994). Cytotoxic T lymphocytes (CTL)

domain (Madu et al., 2007). Madu et al. showed that the infec- arealsocriticalinpreventingdisease(Collissonetal.,2000),and

tivity of Beaudette could be inhibited in the presence of soluble CTLepitopesarepresentonboththespikeproteinaswellasinthe

heparin or in cell lines lacking heparan sulfate, and concluded nucleocapsid(Collissonetal.,2000;Liuetal.,2012).

that heparin sulfate had a role as attachment and entry factor Severaldifferentstrategiesarecurrentlyemployedtogenerate

for Beaudette. Replacing the proposed heparan sulfate binding vaccines against IBV. The strategies employed to evoke protec-

sequence (RRKR/S) in S2 by the corresponding sequence of M41 tiveimmuneresponsesinchickenscanbesubdividedintheuse

(PRRR/S),however,suggestedthattheheparansulfatebindingsite of live attenuated vaccines, viral vectors and DNA plasmids, and

isnotcrucialforentry,asinfectiousvirusescouldberecoveredfrom recombinantIBVs.Manyofthecommerciallyavailablevaccinesto

Verocells(YamadaandLiu,2009).Usingourspikehistochemis- protectpoultryagainstinfectiousbronchitisinthefieldareofthe

tryassaywedemonstratedthatsitewasalsonotrequiredforthe live-attenuatedtype(SjaakdeWitetal.,2011),whilemostofthe

attachmentofBeaudettespikeectodomaintosusceptiblechicken other approaches are still under experimental investigation. The

tissues(Promkuntodetal.,2013)(Fig.8). overviewbelowisgiveninthecontextofthespikeproteinandits

roleinprotection.

6.5. Hostproteases

7.1. Live-attenuatedvaccines

Furinisawell-knownhostproteaseforcoronaviruses(Masters

and Perlman, 2013). Furin-dependent cleavage of the IBV spike Live-attenuatedIBVvaccines,obtainedbyserialpassagingofan

between S1 and S2 is believed to occur during virus assembly IBVisolateinembryonatedeggs,wereamongthefirstIBVvaccines

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Fig.8. Beaudettespikeectodomainbindingtochorioallantoicmembranetissue.SpikehistochemistrywasperformedusingrecombinantwildtypeBeaudettespike

ectodomains(Beau.ED),ortheectodomaininwhichtheproposedheparansulfatebindingsiteSHRKHS(aa686–691)wasreplacedbythesequenceofM41SPRRRS

(Beaud.ED*M41);Beau.EDinthepresenceof10mg/mlheparin,orapplicationofBeau.EDaftertreatmentoftissueswith1mUneuraminidaseofVibriocholerae(VCNA,

asdescribedinPromkuntodetal.,2013).TheproposedheparanbindingsequencewasnotrequiredforbindingofBeaudettespike,norcouldbindingbeblockedwith
he parin;bind in gtoCAMwas de pen dento nsia licacids.S calebars represen t50(cid:2)m.

developedandstillconstitutemostofthecurrentlymarketedIBV roleofthespikeandothergenesinattenuationandinductionof

vaccines.Attenuationisbasedontheadaptationofavirulentfield protectiveimmuneresponses.VaccinationwithrecombinantIBV

isolatetochickenembryos,withtheaimofgeneratingviruseswith Beaudette with spikes of virulent M41 (BeauR-M41(S); Hodgson

reducedvirulenceinchickens,whileretainingtheabilitytoinduce etal.,2004)and4/91(BeauR-4/91(S);Armestoetal.,2011)pro-

sufficientimmuneresponsestoprotectagainstachallengevirus.In tected chickens against a challenge with IBV M41 and -4/91,

attenuatedvirusstrains,mostmutationsareobservedinthespike confirming the importance of homologous spike proteins in the

andintheregionencodingforthereplicaseproteins(Ammayappan induction of protective immune responses. Moreover, the pres-

etal.,2009;MondalandCardona,2004).Itis,however,yetimpossi- enceoftheS1domainofIBVvaccinestrainH120inaBeaudette

bletopredictbasedonageneticsequencewhethertheIBVstrainis backgroundbonewasalreadysufficienttosignificantlydecrease

sufficientlyattenuated,whilepotentenoughtoinduceandeffective morbidity after challenge with the serologically related virulent

immuneresponse.Inaddition,theuseofattenuatedlive-vaccines M41(Weietal.,2014).TherecombinantvirusinducedIBV-specific

posesariskofresidualpathogenicityassociatedwithvaccineback- antibodiestoasimilarextendastheliveattenuatedH120vaccine,

passageinflocks(Abroetal.,2012;McKinleyetal.,2011;OIE,2013). andcouldresultinfewerhistopathologicalchanges.Recombinant

Despitethis,liveattenuatedIBVvaccinesarestillthegoldenstan- IBVsasvaccinesarepromising,asincontrasttovectorvaccinesand

dardtoprotectchickensfromIBV,asimmuneresponsesarehigh. DNAplasmidstheygaincomparablelevelsofprotectionasobtained

by live-attenuated vaccines. Future research of these recombi-

7.2. ViralvectorsandDNAplasmids nantvaccineswilllikelyfocusonthequantificationofprotective

immuneresponsesandtheabilitytoinduceprotectionagainstmul-

DeliveryofspikeproteinsthroughtheuseofDNAplasmidsor tipleserotypes.

viralvectorstochickenshasresultedinvariablelevelsofprotec-

tiveimmunity(Cavanagh,2007).DNAvaccinestudiesshowthat

theimmunizationofchickenswithgenesencodingmultiplestruc- 8. Summarizingconclusions

turalproteinsofIBVcanincreasethelevelofhumoralandcellular

responses compared to the use of individual genes (Guo et al., Inthisreviewwesummarizedthecurrentknowledgeonavian

2010;Yangetal.,2009).Incontrasttovectorvaccinescommonly coronavirus spike proteins. We addressed both the fundamental

used in the poultry field to transport and present parts of other spike characteristics, and provided an overview of experimental

avianpathogens(includingNewcastlediseasevirus,fowlpoxvirus, dataontheroleofthespikeinbindingandentryincellculture,

and herpes virus of turkeys/Marek’s disease virus), no such vac- tissuetropismexvivo,pathogenesisandprotectioninvivo.

cine is marketed for IBV. Delivery of the spike using attenuated Thefundamentalcharacteristicsofthespikeproteinsofavian

strainsofotherpathogens,includingMycoplasmagallisepticumvac- coronaviruses resemble that of other members of the subfamily

cinestrain(Shiletal.,2011)andrecombinantbaculoviruses(Zhang Coronavirinae in many ways. However, the serotype diversity

etal.,2014)showedonlypartialprotectionagainstchallengewith caused by the sequence diversity between S1 proteins seems to

homologousIBV.Vectorvaccinesmightbeimprovedbyadditionof beuniqueforIBV.Coronaviruseswithdivergentspikesequences

immune-relatedmolecules,asthesecanreduceclinicalsymptoms havealsobeenidentifiedinotherpoultryspecies,includingturkey.

andviralRNAlevels(Chenetal.,2010;Shietal.,2011;Wangetal., WhileIBVisprimarilyarespiratorypathogeninchicken,coron-

2009;Zeshanetal.,2011).Importantly,whencloningintoaDNA aviruses of turkey cause predominantly gastrointestinal disease.

plasmidorviralvector,theparticularsequenceoftheS1domain The pathogenesis and tropism of gammacoronaviruses in many

hastobetakenintoaccount,asalreadyoneaminoacidmutation other bird species is less clear, and it remains to be determined

canreducetheabilitytoconferprotectionagainstchallengecon- whether these are truly different viruses or whether IBV has a

siderably(Toroetal.,2014a,b).Whilemoststudiesonlyexpressthe broaderspeciestropismthanpreviouslythought.

S1domain,recentlyToroandcoworkersshowedthattheS2pro- The N-terminal domain of the spike protein contains the

teincanalsocontributetotheprotectionagainstheterologousIBV receptorbindingdomain.ThisdomainofS1oftheprototypeMas-

challenge(Toroetal.,2014a,b).Despitepromisingdevelopments, sachusettsM41strainisbothsufficientandrequiredforattachment

viral vector vaccines and DNA vaccines for IBV still perform less tochickentissues.ThebindingpreferenceandavidityoftrimericS1

wellthanexistinglive-attenuatedvaccines. forchickentissuescorrelateswiththereportedtropismandpath-

ogenicity in vivo. Binding characteristics of recombinant dimeric

7.3. Recombinantviruses S1proteinsdifferinthisrespect,butalsorequirealpha2,3-linked

sialic acids on the cell surface to attach. It remains to be estab-

Studiesinwhichrecombinantvirusesweretestedfortheirabil- lishedwhetherhost-ororgan-specificexpressionofsialicacidsor

itytoprotectagainstchallengeinvivohaveshedmorelightonthe yetunidentifiedhostfactors,includingaputativeproteinreceptor

Please cite this article in press as: Wickramasinghe, I.N.A., et al., The avian coronavirus spike protein. Virus Res. (2014),

http://dx.doi.org/10.1016/j.virusres.2014.10.009
G Model
ARTICLE IN PRESS
VIRUS-96422; No.ofPages12

10 I.N.A.Wickramasingheetal./VirusResearchxxx(2014)xxx–xxx

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understanding on, andtoc om etoanov elc lassifica tio nandnom en- le sions duetoa via ninfec tiousbronchitis viru sinchicksuninoculated and pre-
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Please cite this article in press as: Wickramasinghe, I.N.A., et al., The avian coronavirus spike protein. Virus Res. (2014),

http://dx.doi.org/10.1016/j.virusres.2014.10.009