2016 Altered pathogenicity of a tl_CH_LDT3_03 genotype infectious bronchitis coronavirus due to natural recombination in

2016·1.95 MB·English

VirusResearch213(2016)140–148

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Virus Research

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

Altered pathogenicity of a tl/CH/LDT3/03 genotype infectious
(cid:2)
bronchitis coronavirus due to natural recombination in the 5 - 17kb

region of the genome

ZongxiHan,TingtingZhang,QianqianXu,MengyingGao,YuqiuChen,QiulingWang,
YanZh ao,Yu haoShao ,Huixi nLi,Xian gang Kong,She ngw angLiu ∗

DivisionofAvianInfectiousDiseases,StateKeyLaboratoryofVeterinaryBiotechnology,HarbinVeterinaryResearchInstitute,TheChineseAcademyof

AgriculturalSciences,Harbin150001,People’sRepublicofChina

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

Articlehistory: Aninfectiousbronchitiscoronavirus,designatedasck/CH/LGX/130530,wasisolatedfromanIBVstrain
Receiv ed8October2015 H1 20-vaccina tedchicken inthisstudy .Analysisof th eS1geneshowedtha tisol ateck/CH /LGX /13 053 0was
R19ecNeoivveedm inb errev2i0s1e5d form a tl/CH/LDT3/03-l ike viru s, w ith a nucl eotide se qu en ce simil arity of 9 9%. Howev er, a complete geno mic
sequenceanalysisshowedthatck/CH/LGX/130530wasmorecloselyrelatedtoaMassachusettstype
Accepted19November2015
strain(95%similaritytostrainH120)thantothetl/CH/LDT3/03strain(86%),suggestingthatrecombina-
Availableonline23November2015
tionmighthaveoccurredduringtheoriginofthevirus.ASimPlotanalysisofthecompletegenomic

sequence confirmed this hypothesis, and it showed that isolate ck/CH/LGX/130530 emerged from a
Keywords:
recombinationeventbetweenparentalIBVH120strainandpathogenictl/CH/LDT3/03-likevirus.The
Infectiousbronchitiscoronavirus
Recombin ation results obtained from the pairwise comparison and nucleotide similarity showed that the recombination
Serotype breakpointwaslocatedinthensp14geneatnucleotides17055–17083.InlinewiththehighS1gene

Pathogenicity sequencesimilarity,theck/CH/LGX/130530isolatewasserotypicallyclosetothatofthetl/CH/LDT3/03

Vaccination-challengetest strain(73%antigenicrelatedness).Furthermore,vaccinationwiththeLDT3-Avaccine,whichwasderived

fromthetl/CH/LDT3/03strainbyserialpassaginginchickeneggs,providedgoodprotectionagainst

challengewiththetl/CH/LDT3/03strain,incontrasttothepoorprotectionofferedwiththeH120vac-

cine.Interestingly,isolateck/CH/LGX/130530exhibitedlowpathogenicitytowardspecific-pathogen-free

chickenscomparedwiththenephropathogenictl/CH/LDT3/03strain,whichwaslikelyduetonatural
recombin ationinth e5(cid:2)1 7-kb regionofthegeno me.Ourresult salsoin dicate that there plica se geneof

IBVisolateck/CH/LGX/130530isassociatedwithviralpathogenicity.

©2015ElsevierB.V.Allrightsreserved.

1. Introduction polyprotein by a −1 ribosomal frame-shifting mechanism. ORF1

encodes15non-structuralproteinsassociatedwithRNAreplication

Infectiousbronchitisvirus(IBV),amemberoftheGammacoron- andtranscription.TheIBVgenomeencodesfourmajorstructural

aviridae, order Nidovirales, is an important pathogen that infects proteins:thespike(S)glycoprotein,thesmallenvelope(E)protein,

domestic chickens of all ages, causing an acute, highly conta- themembrane(M)glycoprotein,andthenucleocapsid(N)protein

gious respiratory disease (Cavanagh and Gelb Jr., 2008). The IBV (CavanaghandGelb,2008).Inaddition,IBVhasothergenesthat

genomecontainsasinglepositive-strandRNAmolecule,whichis encodeforproteinsinterspersedamongstructuralgenes,namely
about 27 .6kb lon g and w hich has a cap at its 5(cid:2) end an d a po ly 3a,3b,5 a,a nd5b(B oursnelletal. ,1987).
(A) ta il at i ts 3(cid:2) en d (B oursnel l et al ., 19 87 ). T he gen ome o f IBV Num er ous ser otypes hav e b ee n described for IBV thus far, as

comprisestenopenreadingframes(ORFs),andthefirst20kbof frequentpointmutationsoccurintheS1domainoftheSgeneof

the genome is made up of ORF1, which is a replicase gene. The thevirus.AnotherimportantfeatureofIBV,aswellasothercoro-

replicase has two ORFs, 1a and 1b, and 1b is produced as a 1ab naviruses, is the high rate of homologous and non-homologous

RNA–RNA recombination that has been demonstrated to occur

among selected and unselected markers during the course of

infection (Masters, 2006). RNA recombination in coronaviruses
∗
Correspondingauthor.Fax:[phone]. is thought to result from a copy-choice mechanism (Kirkegaard
E-mailaddress: swliu@ hvri. ac.cn (S. Liu).

http://dx.doi.org/10.1016/j.virusres.2015.11.021
0168-1702/©2015ElsevierB.V.Allrightsreserved.
Z.Hanetal./VirusResearch213(2016)140–148 141

andBaltimore,1986). In this scheme,the viralpolymerase,with PCR(RT-PCR)amplificationaspreviouslydescribed(LiuandKong,

its nascent RNA strand intact, detaches from one template and 2004).

resumeselongationattheidenticalposition,orasimilarposition, Inaddition,apathogenicIBVstrain,tl/CH/LDT3/03,andtwovac-

onanothertemplate.Althoughstrongselectivepressuresareable cine strains, H120 and LDT3-A; the LDT3 was derived from the

tocreatetheappearanceoflocalclusteringofrecombinationalhot tl/CH/LDT3/03strainbyserialpassaginginchickeneggs,wereused

spots(Banneretal.,1990),thesitesofrecombinationareconsid- in the virus cross-neutralization and vaccination-challenge tests

ered to be random (Banner and Lai, 1991). Because homologous in this study. The pathogenic strain tl/CH/LDT3/03 and vaccine

recombinationcanoccurbetweengenomicandsubgenomicRNAs, strain LDT3-A were also used for complete genomic sequencing.

withthelatterprovidingasourceofdonorandacceptortemplates Embryo-propagated viral stocks of these viruses were produced
that beco mem orenume ro usasa fu nction of proximity tothe3(cid:2) byinoculatingthevi rusin toemb ryo nated SPFchic keneg gsviathe
end ofthege nome, therateof rec o mbinatio nin creasesfro m the 5(cid:2) alla ntoiccavity ,an dthe infec tiousallantoic flui dwasco llect ed4 8h
to3 (cid:2)en do fthecoro nav irus ge nome,(Masters ,2006). post-inoc ulation asp rev iouslydes cribed(L iuand Ko ng,2004). Th e

Accumulating evidence suggests that recombination is an titersofthefourviruses,ck/CH/LGX/130530,tl/CH/LDT3/03,H120,

inherent component in IBV evolution, and it is believed that andLDT3-A,weredeterminedbyinoculating10-folddilutionsinto

recombination events can occur in the field under the following groupsoffive10-day-oldembryonatedchickeneggs.Themedian

conditions:whenthereareextremelylargenumbersofchickens, embryoinfectiousdose(EID )wascalculatedusingthemethodof
50

especially when kept at high density; when the virus is easily ReedandMuench(1938).

spread; and when there is a co-circulation of serotypes, includ- All experiments were conducted using standard procedures

ingproofofco-infectionwithmorethanoneserotypeinagiven withtheformalapprovaloftheEthicalandAnimalWelfareCom-

flock(Cavanagh,2007).Thecircumstantialevidenceforrecombi- mitteeofHarbinVeterinaryResearchInstitute,China.

nation,derivedfromgenesequencecomparisons,hasshownthat

therecombinationeventshadoccurredaverylongtimeago.How-
2.2. RNAextraction,geneamplification,andsequencing
ever, it was shown that recombination in the S1 domain of the

S protein of the IBV genome is a mechanism that leads to the
GenomicRNAwasextractedfromvirus-infectedallantoicfluid
emergence of new IBV strains, and it could be responsible for
with the RNAiso Plus kit (TaKaRa, Shiga, Japan) according to the
changes in viral pathogenicity and replication in a single gener- manu fact urer’s i nstru ctio ns and s tored at −80 ◦C until f urt her
ation (Hewson et al., 2014). In general, it is difficult to ascertain
use. Twenty overlapping primers (Zhang et al., 2015) were used
whateffectrecombinationhasontheserotype,pathogenicity,vir-
to amplify the genomes of the isolate ck/CH/LGX/130530, strain
ulence,andtissuetropismofrecombinantstrainsusingonlygene
tl/CH/LDT3/03andtheLDT3-Avaccinestrain.Allgenefragments
sequenceanalyses.Serotypingandanimalstudies,pairedwithdif-
wereamplifiedusingtheRT-PCRkit(TaKaRa)accordingtotheman-
ferential analyses of genes, genomic sequencing, and phylogenetic ufactu rer’sinst ructio ns. The3(cid:2)/5 (cid:2)-t erminiof thethree IB Vst rains
analyses,willbeessentialtoprovideinsightsintothealteredchar-
weredeterminedaspreviouslydescribed(Zhangetal.,2015)using
acteristics of recombinant IBV strains and their effect on poultry the3(cid:2) /5(cid:2)RACEkit( Ta KaRa)acco rdingtoth emanu fac tur er’sins truc-
health.
tions.TheRT-PCRproductsweresequenceddirectlyand/orcloned

intothepMD18-Tvector(TaKaRa),andthreeindependentclones

weresequencedforeachamplicon.
2. Materialsandmethods

2.1. Epidemiologicalbackgroundandvirusisolation 2.3. Sequenceanalysis

An IBV strain, designated as ck/CH/LGX/130530, was isolated The assembly of contiguous sequences of ck/CH/LGX/130530,

from tracheal swabs of diseased broilers suspected of having an tl/CH/LDT3/03 and LDT3-A was performed with GeneDoc soft-

IBVinfectioninGuangxiprovince,China,in2013.Theflockinves- ware (Ammayappan and Vakharia, 2009). Both the S1 gene and

tigatedinthisstudycontainedabout15,000broilers.One-day-old complete genomic sequences of IBV isolate ck/CH/LGX/130530

chickenswerevaccinatedagainstIBVwiththeliveattenuatedH120 were used for BLAST searching of the National Center for

vaccine,and15-day-oldchickensboostedwithMa5livevaccines. BiotechnologyInformation(NCBI)database.Thetl/CH/LDT3/03and

Some of the chickens showed early clinical signs, including list- partridge/GD/S1/42003strains,aswellastheLDT3-Avaccine,were

lessness,huddling,andruffledfeathers,whentheywere20days usedasonegroupofparentalvirusesforsequencecomparisons.

old. On day 25, some of the diseased chickens began to die, and In addition, Massachusetts type H120 and Mass 41 strains were

grossexaminationsshowedmildtoseveretracheitis,nephritis,and selectedandusedasanothergroupofparentalvirusesforsequence

proventriculitis.Themorbidityratewasabout7%,andthemortality comparisons.Similarityandbreakpointanalysesofthecomplete

ratewas3%.Theclinicalsignsofthechickenstendedtodisappear genomicsequenceofck/CH/LGX/130530werealignedwiththose

graduallyafteraboutday35. oftl/CH/LDT3/03,LDT3-A,partridge/GD/S1/42003,H120,andMass

Tracheal swabs were collected from 30 chickens in the dis- 41,andthemultiplealignmentresultswereintroducedintoSim-
eased flock on day 23 a nd centri fuged at 6000×g for 10 min. Plo tver sion 3.5.1toid entifylike lyrecom bina tionbreakpo ints (Lole

Thesupernatantswerepooledandinoculatedintofive9-day-old etal.,1999).SimPlotanalyseswereperformedusinga1000bpwin-

embryonated specific-pathogen-free (SPF) eggs via the allantoic dowwitha100bpstep.IBVstrainMass41wasusedasthequery

cavity(0.2mlperegg).Theeggswerecandleddaily,andembryos strain.Toconfirmregionsinwhichrecombinationeventsoccurred

deathsthatoccurredwithin24hofinoculationwereconsidered in the genome of the ck/CH/LGX/130530 isolate, pairwise com-

to be non-specific. Allantoic fluids were collected at 96h post- parisonsofthecompletegenomicsequenceofck/CH/LGX/130530

inoculation, pooled together, and clarified by centrifugation at wereperformedwiththoseoftl/CH/LDT3/03,LDT3-A,H120,and
3000×g for 5min. The clear supe rnatant was used for furth er M41, andthenuc leotid esim ilar itiesofthediffe rentcorr espon ding

passaging.Thesampleswereblindpassagedfourtimes,andchar- genefragmentswerecalculated.Inaddition,pairwisecomparisons
acteristic I BV lesions, s uch as dw arfing, st untin g, or curli ng of ofthe sequence ofth e3(cid:2)7.0kbr eg ionofck/ CH/LGX/1 30530were

embryos,wereobservedatthefourthpassagelevel.Allantoicfluids performed with those of partridge/GD/S1/42003, tl/CH/LDT3/03,

oftheinoculatedembryoswerecollectedforreversetranscription- andLDT3-Atoelucidatewhetherthetl/CH/LDT3/03-likesequence
142 Z.Hanetal./VirusResearch213(2016)140–148

of isolate ck/CH/LGX/130530 was derived from the pathogenic Birds in groups 3 and 4 were challenged with 1×105 EID of
50
tl/CH/LDT3/03orattenuatedLDT3-Avaccinestrains. pathogenictl/CH/LDT3/03(Liuetal.,2005).Birdsingroup6were

mock-inoculatedwithsterileallantoicfluidandservedastheneg-

2.4. Confirmationoftherecombinationinthenaturalcondition ativecontrol.Trachealswabsandbloodsampleswerecollectedon

days 4, 8, 12, and 16 post-challenge from all birds. The tracheal

In order to confirm the predictive recombination event that swabswereusedforvirusre-isolation,andthebloodsampleswere

occurred in the genomes of the ck/CH/LGX/130530 had a nat- usedtoexamineantibodiesagainstIBV.

ural origin, and was not a methodological artifact originated

during the inoculation in eggs, five of the 30 tracheal swabs
2.9. Virusre-isolationandantibodydetection
which were used for virus isolation were selected and used for

damenptlliyficwaittihonp roifm theres sRweict5ch(cid:2) (s5i(cid:2)t-eG aTnGdG TflAanTAkiTnTgG sGeTqTuGeTnCceAsT GinCdGeCp-e3n(cid:2)-) The tracheal swabs were centrifuged individually at 6000 × g
andRe c3(cid:2)(5 (cid:2)-CAACA AGCCCTTCCGGTGTAAC-3(cid:2)).Oneoftheampli- for 10 min, and each of the supernatant samples was inoculated
intotwotofive9-day-oldSPFembryonatedeggsviatheallantoic
conwassequenceddirectly.
cavity(0.2mlperegg).Theeggswerecandleddailyuntil7days

post-inoculationandthenexaminedforcharacteristicIBVlesions,
2.5. GenBankaccessionnumbers
suchasdwarfing,stunting,orcurlingofembryos.Allantoicfluid

from two of the inoculated embryos was collected, pooled, and
The complete genomic sequences of IBV isolate
used for RT-PCR amplification as previously described (Liu and
ck/CH/LGX/130530, strain tl/CH/LDT3/03 and the LDT3-A vac-
Kong,2004).Apositivesamplewasrecordedifspecificlesionswere
cine strain were deposited into GenBank (accession numbers
observed and the RT-PCR amplification was positive. Sera were
KP343691,KT852992andKR608272,respectively).
examinedforantibodiesagainstIBVusingacommercialenzyme-

linkedimmunosorbentassay(ELISA)kit(IDEXX,Portland,ME,USA)
2.6. Viruscross-neutralizationtests
accordingtothemanufacturer’sinstructions.

Virus neutralization tests were performed using anti-sera
3. Results
against strains ck/CH/LGX/130530, tl/CH/LDT3/03, and H120 to

determinetheirantigenicrelationship.Forserotyping,reciprocal
(cid:2) virus ne utrali zation tes ts were perfo rme d using con stant (102 3.1. MolecularcharacteristicsoftheIBVck/CH/LGX/130530

isolate,tl/CH/LDT3/03strainandtheLDT3-Avaccine
EID ) viral titers and diluted serum against ck/CH/LGX/130530,
50

tl/CH/LDT3/03,orH120inSPFchickenembryos.Theend-pointof
Each of the sequences of the IBV isolate ck/CH/LGX/130530,
eachserumsamplewascalculatedusingthemethodsofReedand

straintl/CH/LDT3/03andtheLDT3-Avaccinewereassembledinto
Muench(1938).Cross-reactivityRvalueswerecalculatedaccording

acompletegenome.Thesizeoftheck/CH/LGX/130530andLDT3-A
totheformulapublishedbyArchettandHorsfall(1950).

genomeswere27,613,27,670and27,687nucleotides,respectively,
excluding thep oly-Ata ilatthe 3(cid:2)e ndsoft hegenomes. Thegenome
2.7. Pathogenicityto1-day-oldSPFchickens

organizationandarrangementofthethreeIBVstrainsareinthe
orderof:5(cid:2) u ntra nslatedregion (U TR ),open rea dingfr ame 1a /1b
Three groups of 10 1-day-old SPF layer chickens were placed
(ORF1 a/1 b),S,ORF3,M,O RF5,N, and3(cid:2) UTR.
inseparateisolators.Birdsingroups1and2werechallengedby

in traocular/ intranasa lroute a tone-d ay -old w ith10 5 EID oft he The results of the BLAST search showed that the S1 gene
50
of isolate ck/CH/LGX/130530 was closely related to two strains,
isolateck/CH/LGX/130530andstraintl/CH/LDT3/03,respectively.

tl/CH/LDT3/03andpartridge/GD/S1/42003(99%similarity),which
Birds in group 3 were mock inoculated with the same volume

were both isolated in Guangdong province, China, in 2003. This
ofsterileallantoicfluid.Bloodsampleswerecollectedondays4,

result indicated that the IBV ck/CH/LGX/130530 isolate has a
8, 12, 16, 20 and 24 post-challenge from all birds. Chicks were

tl/CH/LDT3/03genotype.Incontrast,theS1geneofthisisolatehad
monitoreddailyforclinicalsigns,suchastrachealrales,nasaldis-

lessthan85%similaritytothatoftheH120vaccinestrain.Interest-
charge,wateryeye,andwheezing.Theclinicalsignsfromallthe

ingly,theresultsoftheBLASTsearchusingthecompletegenomic
birdswerecountedbythreepeopleovera2-minperiod.Morbidity

sequenceshowedthatck/CH/LGX/130530wascloselyrelatedtoa
andmortalitywererecordeddailyfor24daysfollowingIBVchal-

Massachusettstypestrain(95%similaritytoH120strain);however,
lenge.Grosslesionswerealsocarefullyexaminedandrecordedfor

it showed less than 86% nucleotide similarity to Massachusetts
thedeadchickens.Thekidneysofthedeadchickensinfectedwith

H120,indicatingthatarecombinationeventlikelyoccurredinthe
IBVstraintl/CH/LDT3/03weresubjectedtoimmunohistochemistry

genomeoftheck/CH/LGX/130530isolate.
(IHC)forthedetectionofIBVantigenusingmonoclonalantibody

6D10(Hanetal.,2013)directedagainstthenucleoproteinaspre-

viouslydescribed(deWitetal.,2011a,b;Xuetal.,2015). 3.2. ThegenomicsequenceoftheIBVck/CH/LGX/130530isolateis

mosaic
2.8. ProtectionprovidedbyvaccinationofH120andLDT3-A

againstchallengeofisolateck/CH/LGX/130530 Toidentifytherecombinationeventsthatpossiblyoccurredin

thegenomeoftheIBVck/CH/LGX/130530isolate,similarityplots

Sixty one-day-old SPF chickens were divided into six groups. wereperformedusingstrainsH120,tl/CH/LDT3/03I,LDT3-A,and
One-day -old birds in gro ups 1 and 3 w ere vacc inate d w ith 104 partri dge/GD/S14 /2003 asrepr esenta tivesofthetwo maingro ups

EID oftheH120vaccineviatheintranasalroute.Birdsingroup2 ofIBVs,whiletheM41strainservedasaquery.Asillustratedin
50

werevaccinatedwiththesamedoseoftheLDT3-Avaccine.Birds Fig.1A,asimilarityplotidentifiedacrossovereventinthensp14of

ingroups4,5,and6weremockinoculatedwiththesamevolume Gene1region.

ofsterileallantoicfluid.Bloodsampleswerecollectedon4,8,12, Toobtainthepreciseregionsofthepossiblecrossoverpoints

16,and20daysofagefrombirdsinallgroups.At20daysofage, involved in the recombination events, the genomic sequence of
bird sin gro ups 1, 2,an d5w erec ha llen gedwit h1 × 105 E ID of the ck/CH /L GX/ 130530 isolate was car eful ly pairwis e compar ed
50

theck/CH/LGX/130530isolateviatheintraocular/intranasalroute. withthoseoftheH120,M41,tl/CH/LDT3/03andLDT3-Aviruses.As
Z.Hanetal./VirusResearch213(2016)140–148 143

Fig.1. RecombinationanalysisoftheIBVck/CH/LGX/130530isolate.SimilarityplotusingMass41asthequerysequence(A).Thedottedlinesshowthededucedrecombination

breakpoints.Thehollowarrowsshowthedifferentfragmentsandtheircolorsarethesameasthoseoftheparentalviruses.Thenumbersshowthenucleotidepositions

ofthecorrespondingfragmentsinthegenomeoftheck/CH/LGX/130530isolate.Multiplesequencealignmentofthepredictedbreakpointandflankingsequencesamong

IBVMass41,H120,ck/CH/LGX/130530,tl/CH/LDT3/03andLDT3-Astrains(B).Thenumbersontherightofeachalignmentshowthenucleotidepositionsinthegenomeof

eachvirus.Thesequencesofck/CH/LGX/130530arelisted,andonlythenucleotidesdifferingfromthoseofck/CH/LGX/130530aredepicted.Theregionwherethetemplate

switches(breakpoint)havetakenplaceisunderlined.Thedeletednucleotidesareindicatedbya-.GenBankaccessionnumbersareinbold.TheGenBankaccessionnumbers

areMass41(AY851295),H120(FJ888351),andpartridge/GD/S14/2003(AY646283).PercentagesofnucleotidesequenceidentityamongMass41,H120,ck/CH/LGX/130530,

tl/CH/LDT3/03andLDT3-Astrains(C).Thepercentagesofnucleotidesequenceidentityofthecorrespondinggenefragmentsareindicated.
144 Z.Hanetal./VirusResearch213(2016)140–148

bTsoacesfttwgto1tsfioihlhhetl vehufe6v/onora rqooCoent eercan ssaOIwmssvu nwHe kine ,rtleewnp iue td/ueole /rufn aCysrnoLrrusbaceroitde atHDufaicatniaesfhnrd dngae ltt lttT/ eliIt h iei mrisw LtysrtBc3tsltoFe dhehGeaohi t V/iienesroc igteelng0epiXeuvena elsnn a .3 ei3ac/e n tldtLps,tr 11twat i ot(cid:2)s htdos DoshahpBsr73 hmtt esliintlweToora.0a,ntryuh 0 a hb3egtt ang5gw ipdo heitikie te- es3 nnnhnpgtecr Aoeb pnnoh0ce hdmeaoourgt1 iokfr cenhnesci rsce w4etcoo/ms tt e ilatnMhCpp gareicemgn antvaieetiHao dooeessc4oe iit btst ttfin f nr/ln1odththmir iLai/uhevd tlnaf t CoGens/s(is t eheeotoCavTgtHe th dXe L terrthtfeaHsqeaah /d aDrie/ ii benfLflouta1sf/miHa oT oDnlLsinoaec3ttecrc13Dirih. hnln Te0km t el abI2-1aRtehTekc3mn5/mAtlr0rCeai) 3iTae//3 elcni ,c 0CelsHeat / -vta0ck ovsg,0 snP3iHrra kk/e/ cuwan g 3C tLcC(//ptksscigatetF-cCGLRoaoH ec/rhougndiiDHC ab aqnXginie/asetd nnnsHsTL.fueh/i/e ms r te nLG1td3wd2et.a/,ot G giL3p/nh (XotBMnmHsrh0n G 0Xltfcet)e/ogii3 1ht.ra15e Xtfi /cs f a a h21seLTs 33uth1c/s itsh D03e1ra00canlt7 hawhetr a-0k3ot5T 2,,secaacl0 itie5r0e33 i1ho sr oid(5krnpr3n5toa-0 Fnuem mee5hbAa 0t3 li sst agah–i(ite. v0bpeuosssF htn.I1eevioa tiegontis1rondrrn7tagaml ehnu es Cagaca , .wtut0t- adsogticed)et2hhloeesr8.iieniini dAooennaeeksn a3rliiir.nnn)ddygeeecsss-)., CH/LDT3/03,andLDT3-Avaccinestrains. 9425039253082560125727261102644526742 5a5bN 51911050176191509806 CCCCCCC CCCCCCC CCTCCCT TTTTTTT DT3/03,andLDT3-Avaccinestrains.Ofthe21mutations,16werethe andtheck/CH/LGX/130530isolate.Nucleotidepositionscorrespondto
3tl./3C.H I/sL oDlaTt3e/ 0c3k/C H/LGX/1305 30 has the same sero type as hogenictl/ 587249 474 T T A A nictl/CH/L 3-Astrain
Theck/CH/LGX/130530isolatewasserologicallycharacterized 03,pat 624 M 67 C C C T athogeneLDT
twAh irwtehea tshs t eur sapeirndes)d. itEcont eddd-e pptoeairrnmetnsitnawel evtrihereuc saaelnsc tuHigl1ae2tne 0id ca bnrydel ttahlt/eiCoHRne/sLehD dipTa 3na/dm03Mo (nuLgDe nTthc3he- D/S14/20 62443 214 C C C A 4/2003,p thevacci
3(tstS(T1hheP.4a9reeF.ob 3 ccctl8Ikhkye s)/i p/o cC2CmlekaH)H e.tae /ne/IsLtnL sh cGGt koch XX/odeC//n .11H LtS3D3r/eL0a0TrGs55u3tX33-m, /0A0t1h i3asises tg0o ora5clalaki3aint/n0teCse, t e H aw xitns/hhiL dtideGhb v i iXHsatitcs/n1i1 enl 2o R3vc0w0 tev l5sr yapst3l raaur0 at,ee hiwiln saoo ohtg fde lie0aicdnd.th 7ie tc n3io inh ot. ydsatt sitnrc oatae whituneeat drrHsad at1lhmi2za0eet 0530withthoseofpartridge/G 50823743238482434 E 1833633469124 TGG TGG TGG CTT withthoseofPartridge/GD/S1 vemutationswerethesamein measthoseshowninFig.1.
f3bcabwcmicsicvltcicataSenndohhihhkikknnega.eiol5sirer ooe///arciaatdyttTid n.cCCCnTAuwr hcsh oill avan7slle HHHeotitmol neTieen beileil,s n aotfr h finn///ddstenleL hoosu LLLfu eeeaegge(nthstDIi fbGGGesn Bthgendtaee H ba3 vTdte3XXXoenVrdedd ydhitcr,1i3r ip fr s /// h o8edkawm es c2ocd-111wI1 h-uet nBn/h Ahh0na333s0rinCciri sioVdssiti aa nnli000th covH cdvhytott vhdisIed555krika7itn ai Hl /acs sa ea 3e333ccei Lcs(tbs1)kctdtCidctoTnh cn 000G rhnscecme6ioia inaase ,os bnkioX enInps ityun bi B vdrans/ee(nhwlonsa/aIsCte/alFoeiVa Bt1 of e ttC dnsHttwi vral yooaeh3V g cwtleaopHltafp/s/tbdfh3yo0le.e dtteLeC ci /eocese 3 )eg5dae1nscG Lcgc4H,keds,r Befn o3 0wtiD incrX i t/rcr hn/ina)cnto 0m oCeova4vLe/Tai,iSotahi1u firntnvtHeDe s3lw caPtiwh ai3 ppdenltl4dpice/ssnT/Fydltr 0i0 hLe oeri ed lty3 dow.ctg ie5oG3dc hcdv8acl nh,/el Wnan3 w9vthl lpa0et Xa letgiailtise0g listrvndo3deisdei /ecrsc hn e t e1na aiaoa.eH pnc ktsiah c.e wdlnnhy31y gnouhoeenyHas1 I iss0 de-eemnduponpcsel.td2 od c d i5ai i h fwk.rhsIs tt0tmbpchpfiwanfe n3 ilihiirwot go/cocyeicdva 0ooCeoeeccvnshikndd-nseniep tkrrvHroeoattetasi i y en toecaf-esherenocl tn/laoiyoctdahobLnltrhscn nbtsl ehhrts,fDs lpr ivaise Ss gl ann ttwaoa(ai /vlroinTe Py ,ntlFoCgesHonil ngif3ereieaF nlitr ue tHneevae gn1/.,tr luceIoc wd 0slnfpneheBe./oA2goc sth c 3 L3 ct hig e-teeen 0nwusschiDrdilalAe rrcr, eo ilit el kseniieno wyek iTka)rrnltc-tt i.tod w lreoit3ei chridhoFuiecs espl a ntch/L nduaa ,nicsodh0fi ooiitgsDiwtllteinrselhc 3 ln ssvergrastwoyTe swkie aoh hdeivbrttal.sn3gettai eoua iiee iNi nohec-gvnnrtsriootsprrAhnhndddoeeeeeesss--ff. Table1 (cid:2)comparisonsofnucleotidesequencesofthe37.0-kbregionofIBVisolateck/CH/LGX/13Pairwise StrainGenomeposition 2043720741213652164122953230342312823 S 57361985126125732654274831 Partridge/GD/S14/2003TGCGCCTC tl/CH/LDT3/03TGCGCCTC TTCGCTTCck/CH/LGX/130530 ATTTTTCTLDT3-A (cid:2)comparedthenucleotidesequencesofthe37.0-kbregionofIBVisolateck/CH/LGX/130530We inthepathogenictl/CH/LDT3/03andck/CH/LGX/130530strain(ingray);incontrast,onlyfisame inthesequenceoftheIBVLDT3-Avaccinegenome.GenBankaccessionnumbersarethesathose
Z.Hanetal./VirusResearch213(2016)140–148 145

Fig.2. Identificationoftherecombinationfromthecollectedtrachealswabsamplesofthechickensshowingclinicalsigns.Fiveoutof30trachealswabsampleswere
iden tifi eddirectlyby RT -PC Rindividuallyu singp rim ersRec5(cid:2) andRec3 (cid:2),and 3weres ho wn tobepos itive(Sam ples1, 2and 4)(A ).Th e PCR product ionof sample1 was

sequenceddirectlyandswitchsite(inblack)andflankingsequenceswereshown(B).Thesequencesofsample1werecomparedwiththoseofH120andtl/CH/LDT3/03strains.

Thesequencesofsample1arelisted,andonlythenucleotidesdifferingfromthoseofck/CH/LGX/130530aredepicted.Thepredictiveswitchsiteandflankingsequences

weresameasthoseofIBVisolateck/CH/LGX/130530.

Table2
Titers wereobtainedusingreciprocalˇvirusneutralizationtests(dilutedserum,constantvirusdoses).

Virus Serum

ck/CH/LGX/130530 tl/CH/LDT3/03 H120

ck/CH/LGX/130530 337.8 181 <2

tl/CH/LDT3/03 181 181 <2

H120 <2 <2 147

Table3

Pathogenicityto1-day-oldSPFchickens.

Group Morbidity Mortality Antibodyresponsea

4db 8d 12d 16d 20d 24d

ck/CH/LGX/130530 3/10 0/10 0/10 1/10 4/10 9/10 10/10 10/10

tl/CH/LDT3/03 10/10 7/10 0/10 4/6 3/3 3/3 3/3 3/3

Negativecontrol 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10

a Numberthatseroconverted/numberinoculated.
b Daysafte rch allenge.

Table4

Resultsofthevaccination-challengetests.

Groupa Vaccinationstrain Challengestrain Morbidity Mortality Antibodyresponseb Virusrecoveryc

Vaccinated Challenged 4dd 8d 12d 16d

4d 8d 12d 16d 20d 4d 8d 12d 16d 20d

1 H120 ck/CH/LGX/130530 0/10 0/10 0/10 1/10 6/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 9/10 5/10 2/10 0/10

2 LDT3-A ck/CH/LGX/130530 0/10 0/10 0/10 1/10 5/10 9/10 10/10 10/10 10/10 10/10 10/10 10/10 1/10 0/10 0/10 0/10

3 H120 tl/CH/LDT3/03 10/10 2/10 0/10 1/10 5/10 10/10 10/10 10/10 8/8 8/8 8/8 8/8 10/10 8/8 1/8 0/8

4 – tl/CH/LDT3/03 10/10 3/10 – – – – – 0/10 7/7 7/7 7/7 7/7 10/10 7/7 0/7 0/7

5 – ck/CH/LGX/130530 2/10 0/10 – – – – – 0/10 9/10 10/10 10/10 10/10 10/10 10/10 3/10 0/10

6 – – 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10

a One-day-oldbirdsingroups1and3werevaccinatedwith104EID50oftheH120vaccine,whilebirdsingroup2werevaccinatedwiththeLDT3-Avaccine.Twenty-day-old
birds in groups 1 , 2, an d 5 were c h allen g ed wi th 105EID5 0of th e ck/CH/LGX /13 0530 isolate, w hile b irds i n g roups 3 and 4 were chall enge d w ith the IB V isolat e tl/CH/LDT3/03.
Birdsingroup6werenotexposedtoanyvirusesandservedasnegativecontrols.
b N um berth a tsero conv erted/nu m beri noculat ed.
c Twopro cedu reswereusedforvirus recoveryafterchallenge,asdescribedpreviously(Zhangetal.,2015).First,embryosthatwereinoculatedwithindividualnasopha-
ryngeals wabsample swer eobs erv edfor lesions.S econ d,RT-PCRu si ngapairo foligonucle otidep rim er s,N(−) andN (+),wasc ond ucted onRNArec overe dfromthe allantoic

fluidofthesameeggs.Theresultsfromthetwoprocedureswereidentical.Numbersindicatethenumberofbirdsthatwerepositiveforvirusrecovery/numberchallenged.
d D ay saf terch alleng e.
146 Z.Hanetal./VirusResearch213(2016)140–148

Fig.3. Pathogenicityoftheisolateck/CH/LGX/130530toSPFchickens.ThekidneyofSPFchickenwhichwasmockinoculatedwithsterileallantoicfluid(A).Therenal

lesionsofthekidneycausedbyinoculationwithisolateck/CH/LGX/130530(B).Kidneysareswollen,tubulesanduretersaredistended,anduricacidcrystalsarepresent.

RepresentationofthetypeofimmunohistochemicalstainingofIBVseeninthekidneysofchickensmockedinoculatedwithsterileallantoicfluid(C)andinfectedchickens
(D).Imageswer et ake nat5 0× magnification.

fromtherespiratorytractat4daysafterck/CH/LGX/130530chal- natedfrommultiplerecombinationeventsbetweenConnecticut,

lenge,andallbirdswerenegativethereafter(Table4).Thechallenge Massachusetts,andArkansasvaccines(MondalandCardona,2007).

viruswasdetectedinallbirdsatdays4and8post-challengewith InthecourseofourcontinuoussurveillanceofIBVinChina,several

theck/CH/LGX/130530isolate. naturalrecombinantIBVstrainshavebeenisolatedandidentified.

ToevaluatetheabilityoftheH120vaccinetoprotectagainst These IBV strains were shown to have originated from recombi-

tl/CH/LDT3/03, ten H120-vaccinated chickens were challenged nationeventsbetweenfieldandvaccinestrains,differenttypesof

withtheIBVstraintl/CH/LDT3/03.Theresultsdemonstratedthat vaccinestrains,ordifferenttypesoffieldstrains(Liuetal.,2013a,b;

all of the chickens developed clinical signs and that two chick- Liuetal.,2014;Zhangetal.,2015).Someoftherecombinantstrains

ensdiedat5and6dayspost-challenge.Meanwhile,allchickens were shown to have undergone multiple recombination events

were positive for virus re-isolation 4 and 8 days post-challenge, (Liu et al., 2013b; Liu et al., 2014; Zhang et al., 2015). In addi-

suggesting that H120 vaccination could not provide protection tion,otherreportsinChinaalsoconfirmedourresults(Zhaoetal.,

againsttheIBVstraintl/CH/LDT3/03.Inaccordancewithourpre- 2013;Fengetal.,2014;Zhouetal.,2014),indicatingthatrecom-

vious results (Liu et al., 2005), the IBV strain tl/CH/LDT3/03 was binationplayedanimportantroleinthecourseofIBVevolution

highlypathogenictoSPFchickens,asitresultedin100%morbidity in China. In this study, we isolated and identified an IBV isolate,

and30%mortality(Table4).Incontrast,onlyasmallproportionof ck/CH/LGX/130530, that likely originated from a recombination

chickens (20%) developed mild clinical signs at about 4–10 days eventbetweenaMassachusetts-typeH120-likestrainandafield

after ck/CH/LGX/130530 challenge. All challenged chickens sur- tl/CH/LDT3/03-likestrain,asshownbySimPlotanalyses,pairwise

vivedthroughtheentireobservationperiodof25days.Noneofthe comparisons,andsimilaritycomparisonsusingcorrespondinggene

chickschallengedwiththeck/CH/LGX/130530isolateshowedsero- fragments.

conversionat4dayspost-challenge,butantibodiesweredetected Thetl/CH/LDT3/03strainwasfirstisolatedin2003inGuang-

byELISAinnearlyallofthebirdsafter8dayspost-challenge. dong province, China (Liu et al., 2005), which was close to

Guangxi province where ck/CH/LGX/130530 was isolated. The

4. Discussion tl/CH/LDT3/03-likeviruseswerealsoisolatedandshowntobeone
ofthemajortypeso fviruses circu latin ginH12 0-va ccinate dc hic ken

Recombination events between different strains of IBV could flocks in southern China, including Guangxi province, in recent
resultintheemerg enceof severaln ovelstrai ns,and the det ection years (Lu oetal.,2 012;Fe ngetal.,2 014).The company at which
ofsom en ove lstrainsha sc ausedco nside rableco ncer n.It hadbeen ck/CH /LGX /13 053 0was isola ted ha dmore tha na20-yea rh istory
re ported thatt heCan adia nQum vstrainorigi natedfro m are com- ofchickenbreeding ,and ithadu sed theH1 20va c cineagai nstIBV
bination even tbe tweenM assa chu settsa ndArkans as-lik e strains fo rmoreth an15yea rs.I th asb eens how edth attheH1 20vacc ine
(Smati e t al., 2 002). Cal ifornia 99-like virus es presumably origi- stra in ma y pe rsis t in va ri ous intern al organ s of chi ckens for 163
Z.Hanetal./VirusResearch213(2016)140–148 147

daysorlonger(CavanaghandGelb,2008).Therefore,recombina- waspreviouslydemonstratedthattheaccessoryproteingenesof

tioneventsbetweentheH120vaccineandfieldstrainsmayleadto IBVcontributedtovirulence(Shenetal.,2003;Younetal.,2005).

thecreationofanewvirus,suchastheck/CH/LGX/130530isolated However,replacingthecompletereplicasegeneofthepathogenic

inthepresentstudy,althoughwecannotexcludethepossibility IBVM41strainwiththatofanon-pathogenicBeaudette-derived

thatthisisolatewasintroducedfromotherregions.However,previ- gene demonstrated that the IBV replicase gene is a determinant

ousstudiesshowedthatthevirusesoftl/CH/LDT3/03genotypehad ofpathogenicity(Armestoetal.,2009).Thiswasalsoobservedin

beenisolatedinthechickenflocksofthecompany(Lietal.,2012, anothercoronavirus,whichledtothebeliefthatchangeswithin

2013)althoughitwasnottheflockinwhichck/CH/LGX/130530 thereplicasegene,especiallytheRNA-dependentRNApolymerase

hadbeenisolated,implicatingthatrecombinationeventhadbeen thatconstitutesthecoreofviralreplicases,canchangethereplicase

likely occurred in this company. The results showed that H120- fidelity,asinthevastmajorityofcases,thealterationsinreplica-

likesequencesinthegenomeoftheck/CH/LGX/130530isolatehad tionfidelitydecreasedviralfitnessandattenuatedvirulence(Smith

lessthan99.8%similaritytothoseoftheH120vaccine,andsimi- etal.,2014,2015).Ourresultsconfirmedthatthereplicasegene

larly,thetl/CH/LDT3/03-likesequencesoftheck/CH/LGX/130530 wasassociatedwithviralpathogenicityalthoughthisobservation

isolate had about 99.7% similarity to those of the tl/CH/LDT3/03 for the ck/CH/LGX/130530 isolate needs to be verified by future

strain.Theseresultsimplythattherehasbeenarecentrecombina- reversegeneticandanimalstudies.

tionevent,causingnearhomogenizationofcertaingenomicregions

betweentwohighlydivergedviruses.Moreover,theoccurrenceof Acknowledgements

sucheventssuggestsanimportantroleforrecombinationbetween

apathogenicfieldIBVstrainandthevaccinestrain. ThisworkwassupportedbygrantsfromtheChinaAgriculture
If the ex tensi ve use o f th e H 120 va ccine in nearly all Researc hSyst erm (No.CARS- 41- K12),N ation al“T welfth Five-Year”
chick en fl ocks in Ch ina cou ld pr ovide g ood pro tect ion aga inst PlanforS cience& Tech nologySuppor t(2015BA D12B03 ),andSpe-
tl/CH/LD T3/03 ch allenge , this would se rve a s evidence against cial Fun d for Ag ro -scientific R esearch in the Public Inte rest (No.
the aforement ioned conc lusio n that the ck /CH /LGX/1305 30 iso- 201 30303 3).

lateoriginatedfromarecombinationeventbetweenH120-likeand

tl/CH/LDT3/03-likeviruses,astherewouldbeonlyasmallchance
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