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]
Trang 1PCR detection and characterization of type-2 porcine circovirus
Andre L Hamel, Lihua L Lin, Cheryl Sachvie, Elsie Grudeski, andGopiP.S.Nayar
Abstract
Apolymerase chainreaction(PCR)assay wasdevelopedfordetecting porcinecircovirus(PCV).Theassayreadilydetected
type-2PCV(PCV-2)andtype-1PCV(PCV-1).The PCRprimersweredesignedbasedonDNAsequencesconserved inallreported PCVgenomes.Type1PCV andtype2PCV bothproduced438bp amplification products,whichwereeasilyidentifiedand
dif-ferentiated fromoneanotherbyrestrictionfragment length polymorphism (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-ical sampleswereidentifiedbyRFLP 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) fut de'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'epreuvefut applique'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 dediagnostic v'te'rinairepourlesepreuves de routineet la
sur-veillance del'infectionparCVP-2.L'analysedesprofilsobtenusparRFLPpourraits'ave'rerutilepour unecaracte'risationpreliminaire
etuneidentificationdes isolats deCVP,deme^meque pourdese'tudessurl'e'pide'miologiemoleculairedesCVP
(Traduit par docteur Serge Messier)
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 2 PCVis noticeablydistinci
Virology Laboratory,VeterinaryServices,ManitobaAgriculture,545 University Crescent, Winnipeg, Manitoba R3T 5S6.
Addresscorrespondenceand reprint requests to Dr Gopi Nayar,telephone: (204)945-7643; fax: (204) 945-8062; e-mail:gnayar@agr.gov.mb.ca.
Received August 20,1999.
~s y It
Trang 2(. \I 7 8 9) l() 1!1 12 13 14 15 16 17 18 N.I
k
Figure 1 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; lane 3: PCV-2 from porcine tonsil and lung; lane 4: PCV-2 from porcine mesenteric lymph node; lane 5: negative porcine lung and tonsil; lane 6: nested PCR from porcine semen Lanes 7-12 representHinAdigests of PCR amplification products from PCV-1, PCV-2A, B, C, D, and E, respectively Lanes 13-18 represent Rsal digests of PCR amplification products from PCV-1, PCV-2A, B, C, D, and E, respectively Lanes M represent the BRL 50-bp DNA ladder, with the 350-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 2 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)
Congenitaltremors inyoungpigsappearstobeassociated 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 processwascarriedout in1of4
sep-araterooms,whichweredesignatedforspecificuse inorderto
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 priorto use.
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,
usingamodification of thepublishedguanidinethiocyanate(GiTC)
Trang 3Table1 List of sizes ofrestriction enzymefragments for PCR of PCV-1 and PCV-2
PCV-2B 322,79,28,9
PCV-2C 401,28,9 PCV-2D 401,28,9
PCV-2E 322,79,28,9
PCV-2B 187,162,89
PCV-2C 187,162,89
PCV-2D 187,162,89
PCV-2D 236,164,38
PCV-2E 181,164,38,38,17
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, 6
211, 194,15,12,6
211,194,15,12,6 211,194,15,12, 6
211,206,15,6
178, 146,94, 11, 6 242,189,7
242,159,30,7 242,189,7
242,189,7 242,178, 11, 7 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)
PCR amplification
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 1 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 5 times
with thereaction mixtureby up-and-downpipetting action under
Trang 4PCV-2B 1 G
PCV-2C 1 PCV-2D 1 A C
PCV-2E 1 PCV-1 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 3 88 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.T T 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
PCV-1 1080 TT.CCACA .A G.TGTGGTGCA G.G .TA T C.G G G.GTG A.T AATGG C GT
Trang 5119 5 TCCTTTTGTTATTTGGTTGGAAGTAATCAATAGTGGA^OTAGAACAGGTerGTGTGTGAAGTAACGGGAGTGG;TAGGAGAAGGGTTGGGGATTGTATG
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
TrAGA TCT.T GT.A TAT G A A.G GG A GG.GT.GGGG G G GGAG GAACAAAGTCGTCAATATTAAATCTCATCATGTCCACCGCCCAGGAGGGCGTTCTGACTGTGGTAGCCTTGACAGTATATCCGAAGGTGCGGGAGAGGCG
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
A -. 1768
A 1768 G CC CCTA.A GT CGT 1759 Figure 2 Alignment of the nucleotide sequences of PCV-1 (represented by GenBank accession number U49186 [5]) and PCV-2A, B, C, D, and E (reprsented
by GenBank accession numbers AF027217 [4], AF112862, AF109398, AF117753 and AF198399 [this study], respectively) Dots Indicate homologous
sequences, dashes Indicate Insertions Arrows represent the directions and locations of PCR primers and their names beside them Cleavage sites for the following restriction enzymes are Indicated by bold italic text: Hinfi (GANTC), HinPl (GCGC), Kpnl (GGTAC), Msel (TTAA), Rsal (GTAC), and Xbal (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 reactionwasaddedto its
appro-priate tube of the 831F/1268R PCR reactionmix(thiswascarriedout
inthe preheated thermocycler) The thermocyclerprogram was
performed as follows: 95°C for 4 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 5 min at
200C
Detection and restriction fragment length
polymorphism (RFLP) analysis of amplifled products
Gelelectrophoresis and photographywereperformedasdescribed
previously (34) Products of PCR amplificationwere subjected to
RFLPanalysisasfollows: 5pLof eachpositive PCR reaction was
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,the entire
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
sequences weregenerated using the Bachrestcomputer program(38) The Clustalcomputer program(39)wasusedtoalign nucleotide
sequences. The RFLPpatternsobserved with clinical PCR
posi-tivesweregivenan RFLPdesignation according to comparison withthepredicted RFLPmaps(Figure 2, Table I) Subsequently,the
Clustalgenerated multiplesequencealignmentwasused forcreating
aphylogenetictreeusing the fastDNAmlcomputer program(40,41)
Figure1contains some examples of the PCR assay for PCV that wasperformed onroutine clinical specimens and on PK-15 cell
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-2A
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
1295 1295 1295 1295 1295 1280 1395 1395 1395 1395 1395 1380
1495 1495 1495 1495 1495
1480
1595 1595 1595 1595 1595 1577
1689 1689 1689 1689 1689 1677
Trang 6Fr-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 Phyolgenetic tree of published PCV sequences The nucleotide
sequencesfromthe following type-2 circoviruses were compared with one
another (GenBank accession numbers and literature references are given
In parenthesis): 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), and with PCV-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 1 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 2 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 2 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
widelyfoundinpopulationsof diseasedpigsthatdisplayawide
Trang 7TableII 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 7 - 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 5 12 19 - 94.7 94.9 98.4 98.2 97.9 97.2
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
thepresentstudywillproveusefulfor furtherresearch on this virus
Trang 8Considering 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
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