Journal of Virological Methods 98 (2001) 25–31

Differentiation of porcine circovirus (PCV)-1 and PCV-2 inboar semen using a multiplex nested polymerase chain

reaction

Junghyun Kim, Dong Un Han, Changsun Choi, Chanhee Chae *Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology,

Seoul National Uni�ersity, Kyounggi-Do, Suwon 441-744, South Korea

Received 25 January 2001; received in revised form 5 June 2001; accepted 5 June 2001

Abstract

A multiplex nested polymerase chain reaction (PCR) was developed for the detection of and differentiation betweenporcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen sampleswere found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight forPCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzedusing multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cellfractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay wasfound to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection ofand differentiation between PCV-1 and PCV-2 in boar semen. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Polymerase chain reaction; Porcine circovirus; Semen

www.elsevier.com/locate/jviromet

1. Introduction

Porcine circovirus (PCV) was classified recentlyinto a newly recognized virus family, the Cir-coviridae (Meehan et al., 1997). Other members ofthe family include the human TT virus (Miyata etal., 1999), chicken anemia virus and the psittacinebeak and feather disease virus in animals (Ritchieet al., 1989; Todd et al., 1990), as well as several

plant viruses, including the subterranean cloverstunt virus, coconut foliar decay virus, and thebanana bunchy top virus (Rohde et al., 1990;Harding et al., 1993; Boevink et al., 1995). Twotypes of PCV have been detected and character-ized, and were subsequently named PCV-1 andPCV-2 (Allan et al., 1998; Meehan et al., 1998).PCV-1 virus has been recognized as a contami-nant in a porcine cell line (PK-15) for over 25years (Tischer et al., 1974). PCV-2, with less than80% nucleic acid homology with PCV-1, has beenidentified as an agent consistently associated withpostweaning multisystemic wasting syndrome (Al-

* Corresponding author. Tel.: +82-31-290-2736; fax: +82-31-294-4588.

E-mail address: swine@plaza.snu.ac.kr (C. Chae).

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0166 -0934 (01 )00348 -2

J. Kim et al. / Journal of Virological Methods 98 (2001) 25–3126

lan et al., 1998; Hamel et al., 1998; Meehan et al.,1998; Choi and Chae, 1999; Choi et al., 2000).

Venereal transmission of viral diseases is a ma-jor concern in both human and veterinarymedicine. In the swine industry, where artificialinsemination is a common method for improvinga particular gene pool, attention has been focusedon the detection of diseases, such as the porcinereproductive and respiratory syndrome virus(Christopher-Hennings et al., 1995) and the pseu-dorabies virus (Medveczky and Szabo, 1981), inwhich clinical signs are not always seen. Recently,PCV-2 nucleic acid has also been detected insemen samples from healthy and experimentallyinfected boars (Hamel et al., 2000; Larochelle etal., 2000). However, the detection of viruses insemen by conventional methods using cell culturehas proved very difficult to date due to the cyto-toxicity of semen samples (Medveczky and Szabo,1981; Weiblen et al., 1992). There is thereforeinterest in developing alternative methods. Al-though PCV-1 and PCV-2 have previously beendifferentiated using the multiplex polymerasechain reaction (PCR) and in situ hybridization(Larochelle et al., 1999; Ouardani et al., 1999;Nawagitgul et al., 2000; Kim and Chae, 2001a),no one has, as yet, reported differentiation be-tween PCV-1 and PCV-2 in semen using multiplexPCR. One objective of this study was therefore todevelop a multiplex nested PCR test for the detec-tion of and differentiation between PCV-1 andPCV-2 in boar semen. The second was to deter-mine which seminal fractions of naturally infectedboars contain PCV nucleic acid.

2. Materials and methods

2.1. Separation and extraction of DNA fromseminal fractions

A total of 60, 10-month-old boars were selectedat random from 30 swine herds (two boars perherd) for the collection of semen samples. Thesamples were separated into seminal fluid, non-sperm cells, and sperm head fractions, as de-scribed previously (van Engelenburg et al., 1993).To obtain the seminal fluid fraction, 100 �l of

semen was centrifuged at 12 000×g for 30 s in amicrocentrifuge. Two volumes of lysis buffer 1(0.15 M NaCl, 0.75% sodium-N-lauroylsarcosine,1.5 mg of proteinase K [Boehringer Mannheim,Indianapolis, IN, USA] per milliliter, and 10 �g ofsheared salmon sperm DNA per milliliter) werethen added to the supernatant and the sampleswere incubated at 60 °C for 1 h. When the re-maining cell pellet was suspended in 100 �l ofphosphate-buffered saline (PBS), lyzed in buffer 1,and centrifuged, the nonsperm fraction containingnonsperm cells and spermatozoal tails was ob-tained from the supernatant. When the remainingpellet was suspended in 100 �l of PBS and lyzedwith buffer 2 (buffer 1 without salmon spermDNA but with 40 mM dithiothreitol), the spermhead fraction was obtained.

Whole semen or seminal lystates from variousfractions were treated with TRIzol (Gibco BRL,Grand island, NY, USA). Briefly, 500 �l of di-luted (1:20 in PBS, 0.01 M, pH 7.4) whole seminallysates or seminal lysates from various fractions,were vortexed with an equal volume of TRIzol.After the addition of chloroform (300 �l), theDNA in the aqueous phase was precipitated with500 �l of isopropanol for 15 min. The final etha-nol-washed DNA pellet was air dried and thendissolved in 30 �l of high-performance liquidchromatography-grade water.

2.2. Virus isolation

Whole semen (2 ml) was frozen and thenthawed, mixed with 20 ml of Hanks balanced saltsolution, and centrifuged at 40 000×g for 1 h.The supernatant was discarded, and the pellet wasresuspended and vortexed in 1 ml of minimalessential medium plus 4% fetal bovine serum.Confluent monolayers of PCV-free PK-15 cellswere inoculated with 200 �l of the suspension at1:2, 1:20, and 1:40 dilutions in minimal essentialmedium plus 4% fetal bovine serum, and werethen incubated for 5 days. The cultures subse-quently frozen and then thawed after 3 days ofincubation, and 1 ml of the medium and the cellswere transferred into a new well containing freshmaintenance medium. Second passage cultureswere incubated for 5 days. Cultures were fixed in

J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31 27

80% acetone and tested for PCV-1 and PCV-2 byin situ hybridization as previously described (Kimand Chae, 2001a). PCV-free PK-15 cells, kindlyprovided by Dr Keith West of Prairie DiagnosticServices in Saskatchewan, Canada, were used toisolate PCV-1 and PCV-2 from boar semen.

2.3. Primers

For PCV-1, the conventional PCR was per-formed using primers previously described(Larochelle et al., 1999), which amplified a 349-base pair (bp) region from open reading frame(ORF) 1. The forward primer was 5�-TTGCT-GAGCCTAGCGACACC-3� (nucleotide positions1369-1388), and the reverse primer was 5�-TC-CACTGCTTCAAATCGGCC-3� (nucleotide po-sitions 1717-1698). The reverse primer for thenested PCR were designed using a computer soft-ware (Oligo 4.0 program, National Biosciences,Plymouth, MN, USA). A species-specific regionwas chosen. The primers amplified a 317-bp frag-ment that was in the 349-bp region amplified inthe first reaction. The reverse primer was 5�-TGTTCTCCAGCAGTCTTCCA-3� (nucleotidepositions 1685-1666). The forward primer fornested PCR also used the same forward primer ofthe conventional PCR.

For PCV-2, the conventional and nested PCRwere carried out using primers previously de-scribed (Ellis et al., 1999; Kim and Chae, 2001b).The forward primer was 5�-CGGATATTG-TAGTCCTGGTCG-3� (nucleotide positions1095-1115), and the reverse primer was 5�-ACT-GTCAAGGCTACCACAGTCA-3� (nucleotidepositions 1570-1549). The nested primers am-plified a 225-bp fragment that was in the 481-bpregion amplified in the first reaction. The forwardprimer was 5�-GATTGTATGGCGGGAG-GAGT-3� (nucleotide positions 1286-1305), andthe reverse primer was 5�-ATTGACGACTTT-GTTCCCCC-3� (nucleotide positions 1510-1491).

2.4. Polymerase chain reaction

Supernatant (10 �l) containing extracted DNAwas used as PCR templates in the first reaction,and 10 �l of the product was used for the second

reaction. The amplification was performed in a50-�l reaction mixture containing 1.25 mMMgCl2, 1×PCR buffer, 0.2 mM of each dNTP, 1�M of each primer, and 2.5 U of Taq DNApolymerase (Perkin Elmer, Foster City, CA,USA). Both reactions were run in a thermocyclerunder (GeneAmp PCR System 9600, PerkinElmer-Cetus, Norwalk, CT, USA) the same con-ditions, 35 cycles of denaturation at 95 °C for 1min, primer annealing at 65 °C for 1 min, andextension at 72 °C for 1 min. The PCR was endedwith a final extension step at 72 °C for 10 min.

The amplified product was visualized by stan-dard gel electrophoresis of 10 �l of the finalreaction mixture on a 2% agarose gel. AmplifiedDNA fragments of specific sizes were located byultraviolet fluorescence after staining with ethid-ium bromide. Their lengths were verified by adigested lambda DNA standard run simulta-neously. The PCR reactions were performed intriplicates. Control DNA from the referencestrain was included in each reaction.

2.5. Sensiti�ity and specificity assays

Sensitivities of PCV-1 and PCV-2 detectionswere also estimated through serial dilutions ofDNA extracts prepared from semen. In the spe-cificity studies, porcine reproductive and respira-tory syndrome virus, porcine parvovirus, porcineepidemic diarrhea virus, transmissible gastroen-teritis, rotavirus, and classical swine fever viruswere tested independently with both sets ofprimers for PCV-1 and PCV-2.

3. Results

3.1. Optimization of PCR conditions

To improve the specificity and sensitivity of themultiplex conventional and mutiplex nested PCRreactions, various MgCl2 concentrations (1–2mM), primer concentrations (0.4–2 �M), anneal-ing (50–68 °C) and denaturing (94, 95, or 98 °C)temperatures, and cycle numbers (30, 35, 40 or45) were tested. The optimum product yield wasachieved with 1.25 mM MgCl2, 1 �M primer,

J. Kim et al. / Journal of Virological Methods 98 (2001) 25–3128

Fig. 1. Multiplex conventional and nested PCR sensitivity forPCV-1. Ten-fold dilutions of the extracted PCV-1 DNA from10−3 to 10−5 were used. Lane M=100 bp DNA ladder; lane1=conventional PCR for 10−3 dilution; lane 2=conven-tional PCR for 10−4 dilution; lane 3=conventional PCR for10−5 dilution; lane 4=nested PCR for 10−3 dilution; lane5=nested PCR for 10−4 dilution; lane 6=nested PCR for10−5 dilution.

Fig. 3. Detection of PCV from seminal fluid fraction bymultiplex conventional PCR and multiplex nested PCR. LaneM=100 bp DNA ladder; lane 1=positive multiplex conven-tional PCR for PCV-1; lane 2=positive multiplex nested PCRfor PCV-1; lane 3=positive multiplex conventional PCR forPCV-2; lane 4=positive multiplex nested PCR for PCV-2;lane 5=positive multiplex nested PCR for PCV-1 from sam-ple; lane 6=positive multiplex nested PCR for PCV-2 fromsample; lane 7=positive multiplex nested PCR for PCV-1 andPCV-2 from sample.

annealing and denaturing temperatures of 65 and95 °C, respectively, and 35 cycles.

3.2. Sensiti�ity and specificity assays

The multiplex conventional PCR test detectedPCV-1 DNA up to a dilution of 10−3, corre-sponding to 1.9×10−3 TCID50 ml−1 and PCV-2DNA up to a dilution of 10−3, corresponding to2.2×10−3 TCID50 ml−1. The multiplex nestedPCR test, on the other hand, detected PCV-1DNA up to a dilution of 10−5, corresponding to2.3×10−4 TCID50 ml−1 and PCV-2 DNA up toa dilution of 10−5, corresponding to 2.7×10−4

TCID50 ml−1 (Figs. 1 and 2). Neither the outer,nor the inner primers cross-reacted with any ofthe viruses tested in this study.

3.3. Comparison of multiplex con�entional PCRand multiplex nested PCR

A total of 18 (30%) and 30 (50%) of 60 wholesemen samples were found to be positive using themultiplex conventional PCR and multiplex nestedPCR test, respectively (Fig. 3). All 18 samplesfound to be positive by multiplex conventionalPCR were also found to be positive by multiplexnested PCR. Of the 18 samples found to bepositive using the multiplex conventional PCR,one tested positive for PCV-1 only, three forPCV-2 only, and 14 for both PCV-1 and PCV-2.Of the 30 samples that tested positive using multi-plex nested PCR, two tested positive for PCV-1only, eight for PCV-2 only, and 20 for bothPCV-1 and PCV-2. PCV-1 and PCV-2 productsobtained using multiplex nested PCR were se-quenced, and their identities confirmed as PCV-1and PCV-2, respectively (data not shown).

3.4. Detection of PCV from seminal fractions

Of the 22 semen samples that tested positive forPCV-1 using multiplex nested PCR, PCV-1 DNAwas detected in 22 of the seminal fluid fractions,nine nonsperm cell fractions, and four sperm headfractions. Of the 28 semen samples that testedpositive for PCV-2 using multiplex nested PCR,PCV-2 DNA was detected in 28 of the seminalfluid fractions, 11 nonsperm cell fractions, andfour sperm head fractions (Table 1).

Fig. 2. Multiplex conventional and nested PCR sensitivity forPCV-2. Ten-fold dilutions of the extracted PCV-2 DNA from10−3 to 10−5 were used. Lane M=100 bp DNA ladder; lane1=conventional PCR for 10−3 dilution; lane 2=conven-tional PCR for 10−4 dilution; lane 3=conventional PCR for10−5 dilution; lane 4=nested PCR for 10−3 dilution; lane5=nested PCR for 10−4 dilution; lane 6=nested PCR for10−5 dilution.

J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31 29

3.5. Virus isolation

Attempts were made to isolate PCV-1 andPCV-2 from 60 whole semen samples. PK-15 cellcultures were inoculated with pooled semen ho-mogenates from the PCR-positive animals. Aftertwo successive passages and an incubation periodof 5 days, no cytopathic effect was observed.However, in four of these samples (6.7%), PCV-2nucleic acid could be detected in the cytoplasm ofinfected cells using in situ hybridization. In thesefour samples, the presence of PCV-2 was alsodetected, using multiplex conventional PCR, inthe culture supernatants after the second passage.

4. Discussion

The multiplex nested PCR described above al-lows for the detection of and differentiation be-tween PCV-1 and PCV-2 in boar semen. Usingthis test, DNA products with the unique sizecharacteristic for each type of PCV were obtained,and sequencing then confirmed them to be type-specific PCV. Most PCV DNA was detected inthe seminal fluid and nonsperm cell fractions ofthe boar semen samples tested. Although PCVDNA was also found to be present in four of thesperm head fractions investigated, it is possiblethat these fractions may have been contaminatedwith minute amounts of PCV DNA from theseminal fluid or nonsperm cells fractions. Infectedcells from the site of virus replication may alsofind their way into the semen. This might explainwhy in 11 of the 60 semen samples tested, PCV

was detected in both the seminal fluid and non-sperm cell fractions.

Serological studies undertaken in differentcountries have demonstrated that both PCV-1and PCV-2 are prevalent in the swine population(Tischer et al., 1986; Dulac and Afshar, 1989;Edwards and Sands, 1994; Hines and Luckert,1995; Magar et al., 2000) and that PCV may,therefore, be a common DNA virus in pigs. Inaddition, the number of seropositives for PCV-2has been found to be greater than that for PCV-1(Magar et al., 2000), indicating that PCV-2 is themain PCV type circulating in the swine popula-tion. Our results would appear to confirm thatPCV DNA is found frequently in boar semen. Ahigh prevalence of TT virus, a virus in the samefamily as PCV, in human semen has also beenreported (Inami et al., 2000; Matsubara et al.,2000).

The presence of PCR inhibitors in semen speci-mens is a well-known diagnostic problem thatfrequently leads to false-negative results (Kriegeret al., 1991; Coombs et al., 1993). Although theexact mechanism for this inhibition is unknown, itmay be caused by the sperm cell DNA (vanEngelenburg et al., 1993; Wiedmann et al., 1993).In order to prevent this problem occurring in thepresent study, a detergent was used instead ofreducing agents. Reducing agents appear to causechromatin decondensation of sperm DNA (Even-son et al., 1980) which may lead to false-negativePCR results (Christopher-Hennings et al., 1995).Of the 60 nonsperm cell fractions tested, five(8.3%) were found to be negative using the undi-luted sample but tested positive in the diluted

Table 1Detection of PCV from various seminal fractions by multiplex nested PCR

Number of samplesSeminal fractions

Sperm headNonsperm cellSeminal fluid

+ −PCV-1 − 2PCV-2 + − − 6

+ 2PCV-2 −+11−−PCV-1, -2 +

+ + − 5PCV-1, -2+ + + 4PCV-1, -2

J. Kim et al. / Journal of Virological Methods 98 (2001) 25–3130

sample (data not shown), suggesting that dilutingmay allow for the detection of virus in at leastsome semen samples where detection might nototherwise have been possible. The success of thisprocedure may be due to the diluting of inhibitorspresent in the cell fractions.

Another problem with PCR assays is the occur-rence of false-positive results caused by thecontamination of reaction mixtures with amplifi-cation products of a previous run in which thesame primer set was used. However, the consis-tently negative results obtained in this presentstudy from the numerous negative controls usedindicate that such false-positives were not a prob-lem, due to the care that was taken during DNAextraction and in the manipulation of each reac-tion mixture. Limiting the number of samples(eight to ten) in each test, and the physical separa-tion of the working area, also served to furtherminimize the risk of false-positive results as aresult of contamination.

The PCR test described in this study can becompleted rapidly, within 1 working day, whereasvirus isolation requires at least 1 week. In thepresent study, PCV was isolated in cell culturesfrom only four of the PCR positive samplestested. Failure to isolate PCV in semen using sucha method may have been due to the cytotoxicityof semen samples as previously reported (Med-veczky and Szabo, 1981; Weiblen et al., 1992). Itwas determined that a minimal dilution factor of1:20 is needed when testing semen for the presenceof virus, in order to circumvent the problem ofcellular toxicity (data not shown). Furthermore, adistinct cytopathic effect was not observed in thecell culture, therefore requiring further serologicalidentification using either indirect immunofluores-cence or in situ hybridization, in order to finallydetect and differentiate between PCV-1 and PCV-2. As such, the multiplex nested PCR method wasfound to be a more convenient, rapid, and reliablemethod for the routine differential diagnosis ofPCV in semen.

The results of this study, therefore, support theuse of PCR as an alternative method to virusisolation in the detection of and differentiationbetween PCV types in semen. A high prevalenceof PCV in pig semen was also found using the

PCR assay described in this study. This latterfinding suggests that semen may be a significantvehicle for the transmission of PCV-2. Althoughthis particular PCR test cannot determine theinfectivity of PCV in porcine semen, the results ofthis study do indicate that it can be used todetermine whether a particular porcine semensample is free of PCV.

Acknowledgements

The research was supported by Ministry ofAgriculture, Forestry and Fisheries-SpecialGrants Research Program (MAFF-SGRP), andBrain Korea 21 Project, Republic of Korea.

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