The nucleotide sequences of the North American isolates of PCV-2 found in the GenBank nucleotide sequence database were predicted to be PCV-2A (unpub- lished observations) and, based on [r]
(1)PCR detection and characterization of type-2 porcine circovirus
Andre L. Hamel,Lihua L. Lin, CherylSachvie,ElsieGrudeski, andGopiP.S.Nayar
Abstract
Apolymerase chainreaction(PCR)assaywasdevelopedfordetectingporcinecircovirus(PCV).Theassayreadilydetected
type-2PCV(PCV-2)andtype-1PCV(PCV-1).The PCRprimersweredesignedbasedonDNAsequencesconserved inallreported PCVgenomes.Type1PCV andtype2PCV bothproduced438bp amplification products,whichwereeasilyidentifiedand
dif-ferentiated fromoneanotherbyrestrictionfragment lengthpolymorphism (RFLP) analysis.Porcine circoviruswasdetected
in55% (931/1693)ofrandomlytestedpigswith various clinicalsignsandlesions,mostof whichweredifficult to differenti-atefrom those associated withporcine reproductiveandrespiratory syndrome(PRRS).The PCRproductsfrom allpositive
clin-icalsampleswereidentifiedbyRFLP tobeonly PCV-2;DNAtestedbyPCRwasextracteddirectlyfromone or moreoflung,
mesentericormediastinal lymph nodes,and tonsil.Type2 PCVwasalso detectedin 6% (2/34)of DNA extracteddirectlyfrom
semen ofrandomlychosen healthyboars Positive PCR reactions from 554 diseased pigs werecharacterizedby RFLP and
categorized into5 different profiles (A-E),of which 82.8%werePCV-2A(456/554),3.0%werePCV-2B(17/554),9.9% were
PCV-2C (55/554), 1.1% werePCV-2D(6/554),and3.2% werePCV-2E (18/554).Thecomplete genomicnucleotide sequences
ofPCV-2A, B, C, D,andEweredetermined and foundtohave at least 95%homology comparedwithoneanother and withall
other PCV-2 foundin theGenBank database All PCV-2 had less than 76%homologywith PCV-1.This PCR assay will hope-fullybe usefultoveterinary diagnosticlaboratories for routinetestingand surveillance of infection with PCV-2 The RFLP
pro-filing system might be useful for preliminary characterization and identification of PCV isolates and might alsobenefit
studiesonthe molecularepidemiologyof PCV
Resume
Uneepreuveparreactiond'amplificationenchat^neparlapolymerase (ACP)futde'veloppeepourdetecterle circovirusporcin(CVP).L'epreuve
afacilement permisdemettreene'videnceleCVP detype-2 (CVP-2)et leCVP detype-i (CVP-1).Lesamorcespourl'ACPfurent6labore'es
apartirdessequencesconserveesd'ADNretrouveeschez tous les CVPrapportes.Unproduit d'amplificationde 438pairesde basefutobtenu apartirde CVP-1 etCVP-2,etuneanalysedupolymorphismedessegmentsobtenussuiteau traitementavecdes enzymes de restriction
(RFLP)apermisdefacilement identifieretdistinguerles deuxtypesde virus.Lorsquel'epreuvefutapplique'edefa,onale6atoirechez des
porcs avecdessignes cliniquesetdes lesionsvaries, pourlaplupart difficilesa'differencierdeceuxassociesausyndrome respiratoireet
repro-ducteurporcin,55% (931/1693)sesont averespositifspourCVP.L'analyseparRFLPademontrequelesproduits d'amplificationde tous
lese'chantillonspositifs correspondaientauCVP-2.Lese6chantillonsd'ADNe'prouvesparACPfurentextraits directement deun ouplusieurs
desspe'cimenssuivants:poumon,ganglion me'sente'riqueoumediastinal,etamygdale. Chez 6% (2/34)des verratsensante'choisisauhasard,
le CVP-2futd6tecte'a partird'ADNextraitd'echantillondesemence.LacaracterisationparRFLP desproduitsd'ACPobtenus de 554porcs
maladesa permis de lesseparera l'inte'rieurdecinq profils differents (Aa E), re'partiscommesuit82,8 % CVP-2A(456/554), 3,0 %
CVP-2B (17/554), 9,9 % CVP-2C (55/554), 1,1 % CVP-2D (6/554) et 3,2 % CVP-2E(18/554) Apre's e'tablissement dessequences
nucle'otidiques completes des CVP-2A, B, C, D et E, il futde'termine'qu'elles avaient au moins 95 % d'homologie entre elles ainsi
qu'aveclesse'quencesdetouslesautresCVP-2 retrouve'sdans la base dedonne'e deGenBank Tous les CVP-2presentaient moinsde 76 %
d'homologieavecCVP-1 CetteanalyseparACPserautileauxlaboratoires dediagnosticv'te'rinairepourlesepreuves de routineet la
sur-veillance del'infectionparCVP-2.L'analysedesprofilsobtenusparRFLPpourraits'ave'rerutilepourunecaracte'risationpreliminaire
etuneidentificationdes isolats deCVP,deme^meque pourdese'tudessurl'e'pide'miologiemoleculairedesCVP
(Traduit pardocteurSergeMessier)
bases ofDNA in asingle-stranded circle(4-8).Twotypesof PCV have beenidentified andcharacterized, PCV-1 and PCV-2 Type1 Porcine circovirus (PCV) is an emergingdisease virusthat is PCV isnon-pathogenicand is foundassociatedwiththecontinuous
widespreadinswine(1-3).Porcine circovirus isthe smallestknown porcinekidney(PK-15)cell line(ATCCCCL31)thatispersistently DNA virus, containing onlybetween 1760 and 1770 nucleotide infected with the virus (5,9) Type PCVis noticeablydistinci
Virology Laboratory,VeterinaryServices,ManitobaAgriculture,545UniversityCrescent, Winnipeg, ManitobaR3T5S6
AddresscorrespondenceandreprintrequeststoDr.Gopi Nayar,telephone: (204)945-7643; fax:(204) 945-8062; e-mail:gnayar@agr.gov.mb.ca. Received August 20,1999
(2)( \I 7 8 9) l() 1!1 12 13 14 15 16 17 18 N.I
k
Figure Detection of PCV by the 438 bp PCR assay Lane 1: PCV-1 from persistently infected PK-15 cell culture; lane 2: negative control reaction with water;lane3: PCV-2 from porcine tonsil and lung; lane 4: PCV-2 from porcine mesenteric lymph node; lane5: negative porcine lung and tonsil; lane 6: nested PCR from porcine semen Lanes 7-12 representHinAdigests of PCR amplification products fromPCV-1, PCV-2A, B, C, D, and E, respectively Lanes 13-18 represent Rsal digests of PCRamplification products from PCV-1, PCV-2A, B, C, D, and E, respectively Lanes M represent the BRL50-bp DNA ladder, with the350-bp fragment indicated by the arrow in the right margin
from type I PCV, with less than 76% overall nucleotide sequence homology (4,6-8) The nucleotide sequences of PCV-2 strains and PCV-1 are compared with one another in previous reports (4,6-8)
Antibodies to PCV-1 have been found in high percentages of pigherdsinEurope (3,9)and North America (1,2) and have also been
detectedincattle, humans, and mice (10) Type-2 PCV has recently beendetectedby PCR in cattle and bison(11)
Porcine circovirus type2isfound in pigs with one or more clin-ical symptoms such as wasting, unthriftiness, anemia, diarrhea, pneumonia andenlarged lymph nodes (7,8,12-21) Pigs that are inflicted with post weaning multisystemic wasting syndrome (PMWS) are found to have PCV-2 in many, if not most, tissues
(7,8,12-23).Wasting,generalized lymphadenopathy,and
intersti-tialpneumonia arethe most often observed lesions duringpost mortemexamination.Granulomatousinflammation,multinucleate giantcells, and inclusion bodiesareoften revealed bymicroscopic examinationoflymphoidtissues.Porcine circovirus type isoften foundinmacrophages,monocytes,andintheinflammatory
infil-trates in avarietyoforgans(7,8,12,13,15-23) Furthermore, congenital
tremors appears to be caused by inutero fetal infection with
PCV-2(24)
Pigsexperimentallyinfected with PCV-2displayclinical signs and lesionstypicallyassociated with this virus(16,23),and co-infection
ofpigletswith PCV-2 and porcineparvovirus appearstoresultin
wasting disease (25) Pigs infectedwith PCV-2 develop a wide rangeof clinical signs andlesions,manyof whicharedifficultto
dif-ferentiate from thosethatareassociatedwithporcinereproductive and respiratory syndrome (PRRS) (7,8,12,13,15-23) Many ofthe
pathologic features attributedtoPRRSmaybe causedby PCV-2,and
ithasbeenproposedthat the PRRSvirus(PRRSV)bemore
appro-priatelyrenamedto porcinearterivirus (25,26) Inlight ofrecent
applicationsofpowerfulnewmolecularbiologicapproaches,such
asPCR andrepresentationaldifferenceanalysis (RDA),the
guide-linesfordefiningacausalrelationshipbetweenamicrobeanda
dis-ease(Koch'spostulates)mayneedtobe rewritten(27)
Young pigsinfected with PCV-2 often suffer fromdepleted lym-phoidsystemsandimmunodeficiency,whichin turnmakes them
susceptible to a variety of other disease problems (12,13,16,22) Congenitaltremorsinyoungpigsappearstobeassociated with
infec-tionbyPCV-2(24) Manyofthe disease problems related to PCV-2
infectioninpigs occur even in"high health, high security herds," where the strictest possible control measures are followed for
sev-eral other diseases(14,15,23,28-31)
Using PCR, in-situ hybridization and immunohistochemistry, workersindiagnostic and research laboratories often detect PCV-2
inavarietyof organs of pigs with various clinical signs and lesions
(6-8,12-22,24-26,30).On theotherhand, there are a verylimited number ofstudies regarding PCV-1, which so far appears to be
detectedby PCRin anextremely lowpercentageof diseasedpigs (18,19)
Inthe present study, a PCR assay for detecting PCV is described, and was designedand tested for routine use in the veterinary
diagnosticlaboratory ThisPCR assay wasused for detectingand
characterizingPCV-2inDNAextracteddirectly fromavarietyof
tis-suestaken from diseased pigs with various clinicalsigns and lesions, andinDNAextracted directly fromsemenofapparently healthy boars
All laboratory procedures were performed using published guidelinesforqualityassuranceofPCRindiagnostic laboratory
set-tings(32) Eachstageofthe PCR processwascarriedoutin1of4
sep-araterooms,whichweredesignatedforspecificuseinorderto
min-imizetherisk ofcrosscontamination(32) Separatesetsofdesignated gloves, labcoats,micropipettorsandfilterbarriertipswereusedin
eachroomfor allsteps
Nucleic acid purification
Throughout1997,1998and the firsthalf of1999,tissuespecimens
from 1693 pigs withvariousclinicalsignsand lesionsweresubmitted
directlytothe PCRlaboratoryandwerestoredat-70°C priortouse
Mostclinical specimens(over 90%)weresubmitted from diseased
swineherdsinManitobaand theremainingspecimenswere sub-mitted from diseased swine operations inOntario, Quebec, and Alberta Nucleic acidswereextracteddirectlyfromone or moreof the following tissues: lung, mesenteric lymph node, and tonsil,
(3)Table1 List of sizes ofrestriction enzymefragments for PCR of PCV-1 and PCV-2
Enzymea Virus Fragment size(bp) Enzyme
Alul PCV-1 320,48,31,27,9 Kpnl
AGCT PCV-2A 322,79,28,9 GGTACC
PCV-2B 322,79,28,9
PCV-2C 401,28,9 PCV-2D 401,28,9
PCV-2E 322,79,28,9
Ddel PCV-1 241,164,30 Msel
CTNAG PCV-2A 187,162,78,11 TTAA
PCV-2B 187,162,89
PCV-2C 187,162,89
PCV-2D 187,162,89
PCV-2E 276,162
Hinfl PCV-1 269,166 Rsal
GANTC PCV-2A 198,164,38,38 GTAC
PCV-2B 274,164
PCV-2C 274,164
PCV-2D 236,164,38
PCV-2E 181,164,38,38,17
HinPl PCV-1 299,136 Xbal
GCGC PCV-2A uncut TCTAGA
PCV-2B uncut
PCV-2C uncut
PCV-2D uncut
PCV-2E uncut
athe nucleotide sequence of eachenzyme's recognitionsite isgivenunderneath its name
Virus
PCV-1
PCV-2A PCV-2B PCV-2C PCV-2D PCV-2E
PCV-1
PCV-2A PCV-2B PCV-2C PCV-2D
PCV-2E PCV-1
PCV-2A PCV-2B
PCV-2C
PCV-2D PCV-2E PCV-1 PCV-2A PCV-2B PCV-2C PCV-2D
PCV-2E
Fragment size(bp) 333, 102
uncut uncut uncut uncut uncut 341, 94
211,206, 15,
211, 194,15,12,6
211,194,15,12,6 211,194,15,12,
211,206,15,6
178, 146,94, 11, 242,189,7
242,159,30,7 242,189,7
242,189,7 242,178, 11, uncut
333,105 333,105 uncut uncut uncut
method (33),exceptthat all buffered solutionswereusedatpH7.5
Foreach extraction, 1-mm-thick slices of frozentissue(approximately
0.5 g intotal)wereimmediately placedindisposable,ice-chilled
50-mLpolypropylenescrew-captubescontaining7mLof ice-cold GiTCbuffer:4 MGiTC (VWR Scientific, Mississauga,Ontario),
0.2 Msodiumacetate, 0.4% sarkosyl detergent,0.1 M 2-mercap-toethanol and 0.05% antifoamA(Sigma, Mississauga,Ontario) Tubeswerekepton iceandhomogenized for10 s at settingnumber4
(outof10)usinga20-mm-wideprobe (PolytronTissueHomogenizer, BrinkmannInstruments, Mississauga,Ontario).Analiquot of crude
tissuehomogenate (1mL)wasimmediately transferredinto 1.5-mL
microcentrifugetubesforstorage at-70°C Thetissuehomogenates
inGiTCbufferwereextracted with phenol and chloroform and
pre-cipitatedinethanolat-20°Cusing apublished method(34).DNA wasextractedfromsemenaccordingtopublished methods (35,36)
PCRamplification
Primersequences weredetermined using the PRIMER computer program(Primer,Cambridge, Massachusetts, USA) The primers were designed from sequences thatwereconserved among the 16isolatesof PCV-2 and4isolates ofPCV-1 that have beenreported
todate The primer sequences were asfollows:831F (5'-GAATG-GTACTCCTCAACTGCTGTCCCAGC-3')and1268R (5'-CCACTC-CCGTTAATTCACACCCAAACC-3'), which correspond to
nucleotide positions 831-859 and 1268-1242, respectively, of
PCV-2A, GenBank accessionnumber AF027217 (4) ThisPCR
yieldeda438bp amplification product.Forthenested PCRassay, a setofouterprimerswasusedfor the firstPCR,whichwas
per-formed priortothe831F/1268RPCR.Theseouterprimerswere as
follows: Circ-OUT-1 (5'-GGTGGAACTGTACCTTJTTTTGGCCCGC-3')and Circ-OUT-2 (5'-CTCCTCCCGCCATACAATCCCCC-3'),
whichcorrespondtonucleotide positions771-797and1304-1282, respectively, of PCV-2A, GenBankaccessionnumber AF027217 (4) This outerPCR yielded a 534bpamplification product All
primer sequences wereanalyzedusingtheBasicLocalAlignment Search Tool(BLAST[37]).Theoligonucleotideprimers were
pur-chasedfrom a commercial customsynthesis facility (Bio/Can Scientific,Mississauga,Ontario, Canada)
Reaction mixesfor PCR contained thefollowing, made up in
ultrapurewater (Sigma, Mississauga, Ontario): 10 mMTris-HCl (pH9.0 at25°C),50 mMKCl,0.1%Triton X-100, 1.5 mMMgCl2,
0.2 mM each of the4 dNTPs(Boehringer Mannheim, Montreal, Quebec),0.3 Mof eachprimerand10U/mLTaq DNApolymerase (Promega, Madison,Wisconsin,USA) Aliquots ofreactionmixture
(24iiL) weredispensed intoprelabeled, 0.5-mLpolypropylene microcentrifuge tubes and overlaidwith drop (20-30 pL)of light
mineral oil (obtained fromlocal grocery or pharmacy stores and
storedinthe dark awayfromsunlight) Thereactiontubeswere
placed in a heatblock (Heatblock IImodel, VWR Scientific,
Mississauga, Ontario) preheatedto80°C Onemicrolitreof eachDNA
samplewas added to itsPCRreactiontube andmixed times
(4)PCV-2A ACGGATCGACGACCTGCGACCGACAAGCACAAGAGAGACGCCACAAAAGT
PCV-2B G PCV-2C
PCV-2D A C
PCV-2E
PCV-1 G TG .A A.C G.C G
PCV-2A 101 GTTCCCGAATCTCAGCACCAAATCGACCCACCCATGTATTTGTGGGAGTA
PCV-2B 101 T PCV-2C 101 C T PCV-2D 101 T T
PCV-2E 101 T G
PCV-1 88 C C.T G.G AAA .C T T G TGC .A A TTG
PCV-2A 201 GGAGAGAACCCTCGGTCCATTGGAAGAATTATAGGATGATGGGCGTCAAC
PCV-2B 201 C
PCV-2C 201 C A.A A C PCV-2D 201 C .A A C PCV-2E 201 A C.T C
PCV-1 188 A .G .TA T.T T T.G CT G C.G G T C PCV-2A 301 AAACAAGATACGAATAGAATCGAAAGCATATATATTGGTCCACCAGCAGA
PCV-2B 301
PCV-2C 301 .C AC.G A C
PCV-2D 301 G.G G A
PCV-2E 301 G
PCV-1 288 G C C.C C.C C.A.C C.G G GAAC G GA.G.C PCV-2A 401 TACGCATCGGGACTTGAACGATTGGCGTCGGACCCGACTTTAAATCGGGT PCV-2B 401
PCV-2C 401
PCV-2D 401 .C C.T
PCV-2E 401
PCV-1 388 c C.T C TC T T A C TT A G
PCV-2A 501 GTACTTAATACGAATCGACTATGAACAGAAGCTGGGCACGGGGTAACATG PCV-2B 501 C PCV-2C 501 .G .C
PCV-2D 501 .G .C PCV-2E 501
PCV-1 488 G C AGC A C G C T.G G.G CG
PCV-2A 601 CGTATTCGCCGACAAATGACACAAAAGGTGAGTACTGGAAGGTGTTGTAT
PCV-2B 601 PCV-2C 601 PCV-2D 601 PCV-2E 601
PCV-1 588 CCG T G TAGGA C G TAG T.A A.T A T T.G T Circ-OUT-1
PCV-2A 701 TAGCGCGCTGAGTTCGGCGGGTGTTCTGCGAAATAGTGATTCTTTGCCCG
PCV-2B 701
PCV-2C 701 T T T C
PCV-2D 701 G PCV-2E 701
PCV-1 688 T.A T.C C.G.G G.TT T
831F w
PCV-2A 801 ATTATCACACGCCGTGAGTCCTACGTTCACGAAGTTTTGAGTATCTGTTT
PCV-2B 801 PCV-2C 801
PCV-2D 801 GG
PCV-2E 801 C PCV-1 788 T G CCA CA
KpnZ-RsaI HinfX
PCV-2A 901 GGAAGAATGCTACAGAACAATCCACGGAGGAA -GGGGGCCAGTTCGTCACCCTTTCCCCCCCATGCCCTGAATTTCCATATGAAATAAATTACTQAGT
PCV-2B 901
PCV-2C 901
PCV-2D 901 G T PCV-2E 901
PCV-1 888 C GG T.CUC.AA GA .T.AAG.AG.GGA A .C .TG.CCTT .C A
Usel NseI NseI RsaI
PCV-2A 998 CrTTTTT -~ATCACTTCGTAATGGTTTTTATTTTTCATTTAGGGTTTAAGTGGGGGGTCT7TTAAGAT'TAAATTCTCTGAATTGTACATACATGGTTACA
PCV-2B 998 A
PCV-2C 998 A PCV-2D 998 A PCV-2E 998 A
PCV-1 988 GTT A T - . A T GA A A C.GC
HInPl XbaI HinfI
PCV-2A 1095 CGGATATTGTAGTCCTGGTCGTATATACTGTTTCGAACGCAGTGCCGAGGCCTACGTGGTCCACATTTCTAGAGGTTTGTAGCCTCAGCCAAAGCT~GG
PCV-2B 1095 T T
PCV-2C 1095 A T C TA T G.T T
PCV-2D 1095 A T C TA T G.T T
PCV-2E 1095 T .CT G.T T C T
(5)1268R Circ-OUT-2
119 TCCTTTTGTTATTTGGTTGGAAGTAATCAATAGTGGA^OTAGAACAGGTerGTGTGTGAAGTAACGGGAGTGG;TAGGAGAAGGGTTGGGGATTGTATG
1195 TT TCT T
1195 T G.G .T CCCA.G.T.C TT
1195 T G C G T C A.G T A TT G
1195 A C.T
1180 T A G CC .TTG CT G C.T TA C CCTT .G .G Circ-OUT-2
GCGGGAGGAGTAGTTACATATGGGTCATAGGTTAGGGCTGTGGCCTTTGTTACAAAGTTATCATCTAGAATAACAGCAGTGGAGCCCACTCCCCTATCA
.G G G
GT CT GG.A C T.A G
G G A G T C A A G
T CC.A.TG AGGGG G GC.AG A A A T.T.G
CCCTGGGTGATGGGGGAGCAGGGCCAGAATTCAACCTTAACCTTTCTTATTCTGTAGTATTCAAAGGGTATAGAGATTTTGTTGGTCCCCCCTCCCGGGG
.T T T
c T
TrAGA TCT.T GT.A TAT G A A.G GG A GG.GT.GGGG G G GGAG
GAACAAAGTCGTCAATATTAAATCTCATCATGTCCACCGCCCAGGAGGGCGTTCTGACTGTGGTAGCCTTGACAGTATATCCGAAGGTGCGGGAGAGGCG
G T G
T T G
G T .G T
.G.GG C.G.C.G G G T.G.GGTA TA ATT A T .GC G.A.CC.C-.T T.TG GAG.A.A TTCTGTA A
-G
.T G A
A CG.C T.T .TT.T.AA C GGT.T AATAT TCTT.TC G.TT
CGGGGGCGGTGTCTTCTTCTGCGGTAACGCCTCCTTGGATACGTCATAGC-TGAAAACGAAAGAAGTGCGCTGTA AGTATT 1768
. 1768
GTC 1768
A -. 1768 A 1768 G .CC CCTA.A GT CGT 1759
Figure 2.AlignmentofthenucleotidesequencesofPCV-1 (represented by GenBank accession number U49186[5])andPCV-2A, B, C, D, and E (reprsented by GenBank accession numbers AF027217[4],AF112862, AF109398, AF117753 and AF198399[thisstudy],respectively) Dots Indicate homologous
sequences,dashesIndicateInsertions Arrows represent the directions and locations of PCRprimersand theirnamesbeside them Cleavage sites forthe following restrictionenzymes areIndicated by bold italic text: Hinfi (GANTC), HinPl (GCGC), Kpnl (GGTAC), Msel (TTAA),Rsal(GTAC), andXbal (TCTAGA)
the mineral oiloverlay Reaction tubeswerethen promptly
trans-ferred into aprogrammable thermocycler (MJ Research Inc., Watertown, Massachusetts, USA) preheatedto80°C For the nested PCRassay,1pL of theouterPCR reactionwasaddedtoits appro-priate tube of the 831F/1268R PCR reactionmix(thiswascarriedout
inthe preheated thermocycler) The thermocyclerprogramwas
performed as follows: 95°C for min, followed by 35 cycles of 95°C for1min,50°C for 1min,70°C for1min.The final extension step wasfor 10minat 70°C The programended with at
200C
Detectionandrestriction fragment length
polymorphism (RFLP) analysis of amplifledproducts
Gelelectrophoresis and photographywereperformedasdescribed
previously (34) Products of PCR amplificationweresubjectedto
RFLPanalysisasfollows: 5pLof eachpositivePCRreactionwas
digestedin separate30-p.L(final volume)reactionsforeach
restric-tionendonucleaseHinfl, HinPlI, KpnI, MseI andRsaI,accordingto
manufacturers recommendations (New England Biolabs,
Mississauga, Ontario) After incubation for4hat37°C,theentire
30pLwasmixed with 6pL of loading dye solutionand gel elec-trophoresedasdescribed (34)
Nucleotidesequencedetermination and analysis Severaloverlapping PCR productsweresequenced essentiallyas previously described (4) but withsomeminormodifications Before sequencing, 10pgofeachPCR productwaspurified witha com-mercialsilica-based kit, accordingtomanufacturers instructions (QlAquick PCR purification kit, Qiagen Inc., Mississauga, Ontario) Purified PCR amplification productsweresequencedat a
com-mercialfacility (DNA Sequencing Laboratory, University of Guelph, Guelph, Ontario) by the Applied Biosystems Prism dye-terminator dideoxysystem.The RFLPmapsofthe PCV-1 and PCV-2 nucleotide
sequencesweregenerated using the Bachrestcomputerprogram(38) The Clustalcomputerprogram(39)wasusedtoalign nucleotide
sequences The RFLPpatternsobserved with clinical PCR
posi-tivesweregivenan RFLPdesignation according tocomparison withthepredicted RFLPmaps(Figure 2, Table I) Subsequently,the Clustalgenerated multiplesequencealignmentwasused forcreating
aphylogenetictreeusing the fastDNAmlcomputerprogram(40,41)
Figure1contains some examples of the PCR assay for PCV that wasperformed onroutine clinical specimens and on PK-15 cell
(6)Fr-1 Fr-2 PCV-2C PCV-2D PCV-2E
r Tae-1 Tae-2
Can-2 Can-5 Can-3 Can-4 Can-1
us-1
PCV-2A US-2 PCV-1
Figure 3.Phyolgenetictreeofpublished PCVsequences.Thenucleotide
sequencesfromthefollowing type-2circoviruseswerecomparedwithone
another(GenBankaccession numbers and literature referencesaregiven
Inparenthesis): PCV-2A (AF027217[4]),PCV-2B(AF112862[thisstudy]),
PCV-2C (AF109398 [thisstudy]), PCV-2D (AF117753 [this study]),
PCV-2E(AF109399[thisstudy]),Can-1(AF055392[6]),Can-2(AF085695), Can-3(AF086835),Can-4(AF086834),Can-5(AF086836),Fr-1(AF055393
[6]),Fr-2(AF055394[6]),US-1(AF055391[6]),US-2(AJ223185[7]),
Tae-1(AF154679), Tae-2(AF166528),andwithPCV-1(U49186[5]) culture derived PCV-1 AllPCR-positivetestsyieldedamplification product of the expectedsizeof 438 bp Artifact bandswere never
observed inanyof the PCR tests,nor had anyfalsepositiveor
false negative PCRtestsbeen observed (Figure and datanot
shown)
Both PCV-1and PCV-2werebothreadilyamplified bythe PCR
assay(Figure1).The PCRamplification productsweresubjectedto
RFLPanalysis andwerefoundtobeeasily identified and charac-terizedasbeingeither PCV-2orPCV-1(Figure1,TableI)
Diseased pigswererandomly testedbythe PCRassayand55%
(931/1693)werefoundtobepositivefor PCV The diseased pigs had
awiderangeofclinicalsigns and lesions.Avarietyoftissueswas
foundtocontainPCV,whichwasreadilydetectedbyPCRusing nucleic acidsextracteddirectlyfromone or moreoflung,mesenteric
lymphnode, and tonsil.Also,semenspecimens fromhealthyboars
wererandomly tested by the nested PCRassayfor PCV,and 6% (2/34)werefoundtobepositive.ThePCRproductsfrom all PCV-positiveclinical sampleswerecharacterized by RFLP analysis and allweredeterminedtobeonly PCV-2
The useofRFLPanalysis onthePCR amplification products enabled5different RFLP profiles of PCV-2 (AtoE)tobeidentified (Figure 1,Table I) The complete nucleotide sequences of
PCV-2A, B,C,D,andEwerealigned and compared withoneanotherand
with PCV-1 (Figure 2) TableI contains alist of the predicted sizes
ofDNAbandfragments generated byRFLPanalysis of PCR
prod-uctsfrom PCV-1 compared with those from PCV-2A,B,C,D,and E Thebandsizesobserved for the RFLP analysis of PCR amplification products showninFigure1 agreed preciselywith thosepredicted from the nucleotidesequences given in Figure The PCR
ampli-fication products from554 different diseased pigs were
charac-terizedbyRFLPanalysis and categorizedasfollows: 82.8% were
PCV-2A(456/554),3.0% werePCV-2B(17/554),9.9% werePCV-2C
(55/554),1.1% were PCV-2D (6/554) and 3.2% were PCV-2E
(18/554) The2boarsemensamples that were found to be
PCR-positivewereidentified byRFLP tobe PCV-2A
Basedonthemultiplesequencealignments, the completegenomic
nucleotide sequences of PCV-2A,B, C, D, and E were foundto
haveatleast95%overall homology comparedwith oneanotherand
less than76%overall homology withPCV-1 (Table II) The values
listedinTableIIresulted from multiplesequencealignment (data
notshown) of the complete nucleotidesequencesof all16reported
PCV-2 strainscurrently represented inthe GenBank database (which includes the5reported in thepresentstudy) compared withoneanother and with PCV-1 Thesamemultiplesequence
align-ment was subjectedtophylogenetic analysis, which resulted in
thetreediagram displayedinFigure3 Based onsimple visual observation of thisphylogenetictree, one cannoticethefollowing: all PCV-2 strains are moreclosely relatedto one anotherand
areclearlya distinctgroupofviruses comparedto PCV-1; the
12PCV-2strainsfrom North Americaappear tobe closely related
toPCV-2A, B, and E (Figure 3, Table II); the2PCV-2strainsfrom Taiwan (GenBank accessionnumbers AF154679 and AF166528)
appear tobeclosely related to the PCV-2 strainsfrom North
America(Figure3, TableII); PCV-2B, C, andD appear tobemore
similartothe2 PCV-2strains from France (GenBankaccession
numbers AF055393 andAF055394, Figure 3,TableII)
Porcine circovirustype wasreadily detectedbyPCRin55%of randomly tested pigs, which had awiderangeof clinical signs and lesions.All of thePCR-positivetestsfor clinicalsampleswere
identifiedbyRFLPanalysistobeonlyPCV-2.The PCRassaywas
designed and demonstrated toreadily detectboth PCV-1 and PCV-2.Therefore,the incidenceof PCV-1indiseasedpigsappeared
tobeextremely low, aresult whichagreed withrecentreports
(18,19)
Althoughmostearlier studies focusonassociating PCV-2 with
PMWS inpigs(4,6-8,12,13,1S-17,22,23,25,26), the results fromthe presentstudyagreed withmore recent studies where PCV-2is
(7)TableII Identity between nucleotide sequences of PCV-1 and PCV-2s known todate'
PCV-1 PCV-2A PCV-2B PCV-2C PCV-2D PCV-2E Can-1 Can-2 Can-3 Can-4 Can-5 Fr-1 Fr-2 US-1 US-2 Tae-1 Tae-2
PCV-1 76.1 76.1 76.1 75.2 75.7 76.2 75.5 75.6 75.5 75.4 76.1 76.2 76.1 76.0 76.3 76.1
PCV-2A 423 - 99.0 96.7 96.2 97.5 99.4 98.6 98.5 98.8 98.2 95.6 95.6 99.8 99.5 97.6 97.7 PCV-2B 423 18 - 96.7 96.0 97.1 99.0 98.2 98.2 98.4 97.9 95.4 95.5 99.2 99.0 97.2 97.3 PCV-2C 423 58 58 97.3 96.1 96.8 95.9 95.8 95.8 95.8 95.3 95.4 96.8 96.5 96.3 96.4
PCV-2D 438 67 71 48 95.6 96.2 95.4 95.3 95.3 95.1 94.6 94.7 96.3 96.0 95.2 95.4
PCV-2E 429 44 51 69 78 97.4 96.5 96.4 96.7 96.3 94.9 95.0 97.6 97.4 96.9 97.0
Can-1 420 11 18 56 67 46 - 99.0 98.9 98.7 98.6 95.8 95.9 99.5 99.3 97.5 97.6
Can-2 433 25 32 72 81 62 18 - 99.6 99.3 99.7 94.5 95.1 98.7 98.5 96.7 96.8
Can-3 432 27 32 74 83 63 19 - 99.3 99.3 94.9 95.0 98.6 98.5 96.6 96.8
Can-4 433 21 28 74 83 58 23 12 12 - 98.9 94.7 94.9 98.9 98.7 96.8 96.9 Can-5 435 32 37 74 87 65 25 12 19 - 94.7 94.9 98.4 98.2 97.9 97.2
Fr-1 422 78 81 83 95 90 74 97 90 94 94 - 99.8 95.8 95.5 95.5 95.6
Fr-2 421 78 79 81 94 88 72 87 88 90 90 - 95.8 95.5 95.5 95.6
US-1 422 14 56 65 42 23 25 19 28 74 74 - 99.6 97.7 97.9
US-2 424 18 62 71 46 12 27 79 23 32 79 79 97.6 97.7
Tae-1 419 42 49 65 84 54 44 58 60 56 37 79 79 40 42 - 99.5
Tae-2 423 40 47 63 81 53 42 56 56 55 49 77 77 37 40
aTheClustalVcomputer program(19),wasusedtoperform multiple alignmentsof thecompleteviral genomenucleotide sequences Theabove listed percent values for sequencehomology and number ofpolymorphic(different)siteswereobtainedfrom these sequence alignments
The following nucleotide sequenceswere compared (GenBank accession numbers and literature references aregiven in parenthesis):
PCV-1 (U49186 [5]), thefollowing PCV-2's: PCV-2A (AF027217 [4]), PCV-2B (AF112862 [this study]), PCV-2C (AF109398 [this study]), PCV-2D (AF117753 [this study]), PCV-2E (AF109399 [this study]), Can-1(AF055392 [6]), Can-2 (AF085695), Can-3(AF086835), Can-4 (AF086834), Can-5(AF086836), Fr-1(AF055393 [6]),Fr-2(AF055394 [6]), US-1 (AF055391 [6]),US-2(AJ223185[7]),Tae-1(AF154679), Tae-2 (AF166528)
bPercentages of nucleotideidentityarepresented above thediagonal
cNumberof polymorphic sitesareshown below thediagonal Polymorphicsites include insertions
varietyof clinicalsignsand lesions(14,17,18,20,21,25).Inthe pres-entstudy,PCV-2wasdetectedbyPCRinnucleic acids extracted directly fromone or moreoflung,mesentericlymph node, and
ton-sil These observationsagreedwithrecentreportsof PCV-2being foundin avarietyoforgansfrom diseased pigs(7,8,12,13,15-18, 21-23,25).Forthesereasons, since 1997 ourlaboratory has been
rou-tinelyusingthe PCRassay to testpools ofatleastonetissuefrom each diseased pig(4,11,14)
Our observations appeared toagree withrecentreports
con-cerning thewide rangeof clinical signs and lesionscaused by PCV-2, manyofwhich aredifficultto differentiate from those associated with PRRS(7,8,12,13,15,16,18,19,21-23) Further research will berequiredinordertodetermine whetheror notPCV-2causes orexacerbatesmanyof thepathologic features attributedtoPRRS
(23,25)
Circovirusesareveryhardy andcaneasilysurvivemanyharsh environmental conditions(5,9) Basedon ourpreliminary survey of semenfromhealthy boars,werecommend further studies on the
prevalence ofcircovirus insemen Considering the high
preva-lence ofPCV-2 in diseasedpigs and the wide variety of tissues
foundtocontainPCV-2,werecommendthat any studies on pig dis-easesandstudies involvinganypig tissues or cell culture products
would be wise to include testing for PCV-2 by PCR assay and
preferentially by themoresensitive nested PCR
Over80%of the PCV-2strainsdetectedby PCR in diseased pigs wereidentifiedbyRFLPanalysistobe PCV-2A, and nearly10% of
PCV-2strains werefoundtobe PCV-2C TheRFLPanalysis allows laboratoriestocharacterize andidentifyvirus strainsandisoften used for tracking andmonitoringthespread of thevirusandfor
epi-demiological studies(42-44).Further research willberequiredin
ordertodetermine whetheror nottheremaybeanydifferencesin
biologicalbehavior(virulence and pathogenicity) between different field isolates of PCV-2
Thecompletegenomic DNA sequencesfor all known isolates of PCV-2werefoundtohaveover95%homology withoneanother and less than76%homologywith PCV-1.Thenucleotidesequencesof the North American isolates of PCV-2 found in the GenBank nucleotidesequencedatabasewerepredictedtobe PCV-2A (unpub-lishedobservations) and, basedonmultiplesequencealignment and phylogenetic analysis, they appeared to be closely related to PCV-2A.Thiswasoflittlesurpriseconsideringourobservations that
PCV-2Awasthe predominant PCVfoundindiseased Canadian pigs
The nucleotide sequences of theFrench isolates of PCV-2 were
predictedtobePCV-2C and, basedonmultiple sequence alignment
andphylogenetic analysis, they appearedtobe closely related to
PCV-2C andD.The nucleotidesequences of the Taiwanese isolates
of PCV-2 werepredictedtobePCV-2A and, based on multiple sequencealignmentandphylogenetic analysis, theyappeared to be
closely relatedtoPCV-2A.Thesepreliminary findingsmay be of some useforfuture molecular epidemiologicalstudies Hopefully,
the nucleotidesequencesofPCV-2A, B, C, D, and E characterized in
(8)Considering the application of powerfulnew molecular bio-logic diagnostic techniques suchasPCR and RDA, infection of pigswith PCV-2maybe merely"thetipof theiceberg"regarding
emerginginfectiousdiseaseagents The application of these and
othernewtechniquesarelikelytolead to theidentificationof pre-viouslynon-culturable infectious disease agents (27,4547) Until
these previously non-culturable infectiousagents are identified, further studies will be requiredinordertodetermine the role of
PCV-2 indiseasesofpigs
Thecompletegenomic DNA sequencesof PCV-2B,C, D,andE
frompigs were deposited in the GenBank nucleotide sequence
database Their GenBank accessionnumberswere as follows: AF112862 (PCV-2B), AF109398 (PCV-2C), AF117753(PCV-2D), and AF109399 (PCV-2E) The complete genomic DNA sequence of
PCV-2A (GenBankaccession number AF027217) was previously
reported by this laboratory(4)
The PCRassaydescribedin the present study was found to be a
rapid andsensitivetechnique,which might prove useful to
vet-erinarydiagnostic laboratories forroutinetestingand surveillance of PCV infectioninpigs.The describedRFLPsystem, orsimilar sys-tems, are verylikelyto proveusefulfor performing preliminary char-acterizationand identification of PCV isolates and might benefit studiesonthe molecular epidemiology of PCV
We thankthe veterinary pathologists,post mortem tech-nologists and clerical staff of theVeterinaryServices Branch DiagnosticLaboratory for their valuable contributionstothe routine
diagnostic clinicalcase reports
1 Dulac GC, AfsharA.Porcinecircovirus antigens inPK-15cell line(ATCC CCL-33) and evidence of antibodiestocircovirus in
Canadian pigs Can J Vet Res1989;53:431-433
2 HinesRK,LukertD.Porcine circovirus: aserologicalsurveyof
swine intheUnited States SwineHealth Prod 1995;3:71-73
3 TischerI,Geldblom H,VettermannW,HochMA.Distribution of antibodies toporcinecircovirus in swinepopulations of differentbreeding farms Arch Virol 1995;140:737-743
4 Hamel AL,LinLL, NayarGPS Nucleotidesequenceof porcine
circovirusassociated withpostweaningmultisystemicwasting syndromeinpigs.J Virol1998;72:5262-5267
5 Meehan BM, CreelanJL,McNultyMS,ToddD Sequence of porcine circovirus DNA: affinities withplant circoviruses
J Gen Virol1997;78:221-227
6 MeehanBM, McNeilly F, Todd D,etal Characterization of novelcircovirusDNAsassociatedwithwastingsyndromesin pigs JGen Virol1998;79:2171-2179
7 Morozov I,Sirinarumitr T,SordenSD, etal Detection ofa
novel strain of porcine circovirus inpigswithpostweaning multisystemic wastingsyndrome J Clin Microbiol 1998;36:
2535-2541
8 Onuki A, Abe K, Togashi K, Kawashima K, Tanehi A,
Tsunemitsu H Detectionof porcinecircovirusfrom lesions of
apig with wasting diseaseinJapan.J Vet Med Sci 1999;61:
1119-1123
9 Allan GM,PhenixKV,ToddD,McNulty MS Some biological and physico-chemicalpropertiesofporcinecircovirus J VetMed SerB1994;41:17-26
10 TischerI,BodeJ, Timm H, etal.Presenceof antibodiesreacting withporcine circovirus in sera of humans, mice, and cattle Arch Virol1995;140:1427-1439
11 NayarGPS,HamelAL, Lin L,Sachvie C, Grudeski E, Spearman G Evidence ofcircovirus in cattle with respiratory disease andinaborted bovine fetuses CanVet J 1999;40:277-278
12 Allan GM, McNeilly F, Kennedy S,etal Isolationofporcine
cir-covirus-like viruses frompigswithawastingdiseaseinthe USA and Europe J Vet Diag Invest 1998;10:3-10
13 EllisJA,HassardL,ClarkE, etal Isolation ofcircovirusfrom lesions ofpigswithpostweaning multisystemic wasting
syn-drome CanVet J1998;39:44-51
14 NayarGPS,HamelA, Lin L.Detectionand characterization of porcinecircovirusassociated withpostweaningmultisystemic
wastingsyndromeinpigs Can Vet J1997;38:385-386
15 Rosell C, SegalesJ,Plana-DuranJ, etal Pathological,
immuno-histochemical, and in-situhybridization studies of natural
casesof postweaning multisystemicwastingsyndrome(PMWS)
inpigs.JCompPath1999;120:59-78
16 BalaschM,SegalesJ,RosellC,etal Experimental inoculationof
conventional pigs with tissue homogenates frompigs with post-weaningmultisystemicwastingsyndrome J Comp Path
1999;121:139-148
17 Choi C, Chae C In-situ hybridization for the detection of porcine circovirus inpigs with postweaningmultisystemic
wastingsyndrome J CompPathol1999;121:265-270
18 Larochelle R, AntayaM, Morin M, Magar R.Typingofporcine circovirus in clinical specimensby multiplex PCR J Virol Methods 1999;80:69-75
19 Larochelle R, Morin M, Antaya M, MagarR.Identification and incidence ofporcine circovirus inroutinefieldcases inQuebec
asdeterminedbyPCR Vet Rec1999;145:140-142
20 McNeilly F, Kennedy S, Moffett D,etal.Acomparisonofinsitu
hybridization and immunohistochemistry for the detection of
a newporcinecircovirus informalin-fixedtissuesfrompigs with post-weaningmultisystemicwastingsyndrome JVirol Methods 1999;80:123-128
21 Ouardani M, Wilson L, Jette R, MontpetitC,DeaS.Multiplex PCR for detection andtyping ofporcine circoviruses J Clin Microbiol1999;37:3917-3924
22 Ellis J, Krakowka S, LairmoreM,etal.Reproductionof lesions ofpostweaningmultisystemicwastingsyndromeingnotobiotic piglets JVetDiagnInvest1999;11:3-14
23 Harding JCS, Clark EG Recognizing and diagnosing
post-weaning multisystemic wasting syndrome (PMWS) Swine Health Prod 1997;5:201-203
24 StevensonGW,KiupelM, MittalSK,ChoiJ,LatimerKS,Kanitz
CL Distributionofporcine circovirus inneonatalpigs with
congenitaltremors.ConfResWorkAnimDis1999;80:217
25 AllanGM,KennedyS,McNeillyF,etal.Experimental
(9)porcine circovirusandporcineparvovirus.JComp Path 1999; 121:1-11
26 EllisJ,KrakowkaS,AllanG,ClarkE,Kennedy S The clinical scopeofporcinereproductive andrespiratorysyndromevirus
infection has expandedsince 1987: analternative perspective VetPath1999;36:262-265
27 FredericksDN,RelmanDA.Sequence-based identification of microbial pathogens:areconsideration of Koch's postulates Clin Microbiol Rev 1996;9:18-33
28 Clark EG, Harding JC.Porcine circovirusandpost-weaning
mul-tisystemic wasting syndrome ProcAmAssoc Swine Pract 1998;29:445-447
29 Clark EGPost-weaningmultisystemicwastingsyndrome.Proc AmAssocSwinePract1997;28:499-501
30 LeCannP,AlbinaE,Madec F, Cariolet R, JestinA.Piglet wast-ingdisease.VetRec 1997;141:660-661
31 Segales J,Sitjar M, Domingo M, etal First reportof post-weaningmultisystemic wasting syndrome inpigs in Spain Vet Rec 1997;141:600-601
32 Neumaier M, Braun A,Wagener C.Fundamentals ofquality
assessmentof molecularamplificationmethodsinclinical
diag-nostics.Clin Chem 1998;44:12-16
33 ChomczymskiP,Sacchi N.Single-stepmethodof RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extrac-tion.Anal Biochem 1987;162:156-159
34 HamelAL,Wasylyshen MD, Nayar GPS.Rapid detection of bovine viraldiarrheavirusbyusing RNAextracted directly from assorted specimens andaone-tubereversetranscriptionPCR
assay.JClin Microbiol 1995;33:287-291
35 Christopher-Hennings J, Nelson EA,etal.Persistenceof porcine reproductive and respiratorysyndrome virus in serum and
semenof adult boars J Vet Diag Invest 1995;7:456-464
36 Christopher-Hennings J, Nelson EA, Nelson JK,etal Detection of porcinereproductive and respiratory syndromevirus in
boarsemenby PCR J Clin Microbiol1995;33:1730-1734
37 Altshul SF, GishW,Miller W, Myers EW, Lipman DJ.Basiclocal alignmentsearch tool J Mol Biol 1990;215:403-410
38 Fristensky B, Lis JT,Wu R.Portable microcomputer software for nucleotide sequence analysis NucleicAcids Res 1982;10: 6451-6463
39 Higgin DG, SharpP CLUSTAL: apackagefor performing multiplesequencealignmenton a microcomputer.Gene1988;
45:333-338
40 Felsenstein J.1981.EvolutionarytreesfromDNAsequences:A
maximumlikelihood approach J Mol Evol 17:368-376
41 Olsen GJ,Matsuda H, Hagstrom R,OverbeekR.fastDNAml:A
tool forconstruction ofphylogenetictreesofDNA sequences
usingmaximumlikelihood.Comput Appl Biosci 1994;10:41-48
42 Andreyev VG, Wesley RD,Mengeling WL,Vorwald AC, Lager KM Geneticvariation and phylogenetic relationships of
2porcinereproductive andrespiratorysyndromevirus(PRRSV) fieldstrainsbasedonsequenceanalysisofopenreading frame
5.ArchVirol 1997;142:993-1001
43 Gagnon CA, DeaS Differentiation between porcine
repro-ductiveandrespiratorysyndromevirusisolatesby restriction fragment length polymorphismof their ORFs6and7genes.Can
J Vet Res1998;62:110-116
44 WesleyRD,MengelingWL, LagerKM,Clouser DF,Landgraf JG, FreyML.Differentiation ofaporcinereproductive and respi-ratory syndromevirus vaccine strainfrom NorthAmerican
field strainsby restriction fragment length polymorphism analysisof ORF J Vet Diagn Invest 1998;10:140-144
45 RelmanDA.The search for unrecognized pathogens Science 1999;21:1308-1310
46 RelmanDA Emerging infections andnewly-recognized pathogens.Neth J Med 1997;50:216-220