Journal of Virological Methods 98 (2001) 25 – 31

www.elsevier.com/locate/jviromet

Differentiation of porcine circovirus (PCV)-1 and PCV-2 in

boar 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 Uni6ersity, 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 between
porcine circovirus (PCV)-1 and PCV-2 in boar semen. Eighteen (30%) and 30 (50%) out of 60 whole semen samples
were found to be positive for PCV using multiplex conventional PCR and multiplex nested PCR, respectively. Of the
30 positive samples obtained using multiplex nested PCR, two were found to be positive for PCV-1 only, eight for
PCV-2 only, and 20 for PCV-1 and PCV-2. When the separated fractions of PCV-contaminated semen were analyzed
using multiplex nested PCR, PCV DNA was found to be present mainly in the seminal fluid and nonsperm cell
fractions. When compared with the virus isolation method commonly used to detect viruses, this PCR assay was
found to be more sensitive and rapid and, as such, may prove to be a good alternative method for the detection of
and 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

1. Introduction plant viruses, including the subterranean clover

Porcine circovirus (PCV) was classified recently
into a newly recognized virus family, the Cir-
coviridae (Meehan et al., 1997). Other members of
the family include the human TT virus (Miyata et
al., 1999), chicken anemia virus and the psittacine
beak and feather disease virus in animals (Ritchie
et al., 1989; Todd et al., 1990), as well as several
* Corresponding author. Tel.: + 82-31-290-2736; fax: + 82-
31-294-4588.
E-mail address: swine@plaza.snu.ac.kr (C. Chae).
stunt virus, coconut foliar decay virus, and the
banana bunchy top virus (Rohde et al., 1990;
Harding et al., 1993; Boevink et al., 1995). Two
types of PCV have been detected and character-
ized, and were subsequently named PCV-1 and
PCV-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 25
years (Tischer et al., 1974). PCV-2, with less than
80% nucleic acid homology with PCV-1, has been
identified as an agent consistently associated with
postweaning multisystemic wasting syndrome (Al-

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 3 4 8 - 2
26 J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31
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 veterinary
medicine. In the swine industry, where artificial
insemination is a common method for improving
a particular gene pool, attention has been focused
on the detection of diseases, such as the porcine
reproductive and respiratory syndrome virus
(Christopher-Hennings et al., 1995) and the pseu-
dorabies virus (Medveczky and Szabo, 1981), in
which clinical signs are not always seen. Recently,
PCV-2 nucleic acid has also been detected in
semen samples from healthy and experimentally
infected boars (Hamel et al., 2000; Larochelle et
al., 2000). However, the detection of viruses in
semen by conventional methods using cell culture
has proved very difficult to date due to the cyto-
toxicity of semen samples (Medveczky and Szabo,
1981; Weiblen et al., 1992). There is therefore
interest in developing alternative methods. Al-
though PCV-1 and PCV-2 have previously been
differentiated using the multiplex polymerase
chain 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 multiplex
PCR. One objective of this study was therefore to
develop a multiplex nested PCR test for the detec-
tion of and differentiation between PCV-1 and
PCV-2 in boar semen. The second was to deter-
mine which seminal fractions of naturally infected
boars contain PCV nucleic acid.
2. Materials and methods
2.1. Separation and extraction of DNA from
seminal fractions
A total of 60, 10-month-old boars were selected
at random from 30 swine herds (two boars per
herd) for the collection of semen samples. The
samples 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 ml of
semen was centrifuged at 12 000× g for 30 s in a
microcentrifuge. 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 mg of
sheared salmon sperm DNA per milliliter) were
then added to the supernatant and the samples
were incubated at 60 °C for 1 h. When the re-
maining cell pellet was suspended in 100 ml of
phosphate-buffered saline (PBS), lyzed in buffer 1,
and centrifuged, the nonsperm fraction containing
nonsperm cells and spermatozoal tails was ob-
tained from the supernatant. When the remaining
pellet was suspended in 100 ml of PBS and lyzed
with buffer 2 (buffer 1 without salmon sperm
DNA but with 40 mM dithiothreitol), the sperm
head fraction was obtained.
Whole semen or seminal lystates from various
fractions were treated with TRIzol (Gibco BRL,
Grand island, NY, USA). Briefly, 500 ml of di-
luted (1:20 in PBS, 0.01 M, pH 7.4) whole seminal
lysates or seminal lysates from various fractions,
were vortexed with an equal volume of TRIzol.
After the addition of chloroform (300 ml), the
DNA in the aqueous phase was precipitated with
500 ml of isopropanol for 15 min. The final etha-
nol-washed DNA pellet was air dried and then
dissolved in 30 ml of high-performance liquid
chromatography-grade water.
2.2. Virus isolation
Whole semen (2 ml) was frozen and then
thawed, mixed with 20 ml of Hanks balanced salt
solution, and centrifuged at 40 000× g for 1 h.
The supernatant was discarded, and the pellet was
resuspended and vortexed in 1 ml of minimal
essential medium plus 4% fetal bovine serum.
Confluent monolayers of PCV-free PK-15 cells
were inoculated with 200 ml of the suspension at
1:2, 1:20, and 1:40 dilutions in minimal essential
medium plus 4% fetal bovine serum, and were
then incubated for 5 days. The cultures subse-
quently frozen and then thawed after 3 days of
incubation, and 1 ml of the medium and the cells
were transferred into a new well containing fresh
maintenance medium. Second passage cultures
were incubated for 5 days. Cultures were fixed in
 J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31
80% acetone and tested for PCV-1 and PCV-2 by
in situ hybridization as previously described (Kim
and Chae, 2001a). PCV-free PK-15 cells, kindly
provided by Dr Keith West of Prairie Diagnostic
Services in Saskatchewan, Canada, were used to
isolate 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 positions
1369-1388), and the reverse primer was 5%-TC-
CACTGCTTCAAATCGGCC-3% (nucleotide po-
sitions 1717-1698). The reverse primer for the
nested PCR were designed using a computer soft-
ware (Oligo 4.0 program, National Biosciences,
Plymouth, MN, USA). A species-specific region
was chosen. The primers amplified a 317-bp frag-
ment that was in the 349-bp region amplified in
the first reaction. The reverse primer was 5%-
TGTTCTCCAGCAGTCTTCCA-3% (nucleotide
positions 1685-1666). The forward primer for
nested PCR also used the same forward primer of
the conventional PCR.
For PCV-2, the conventional and nested PCR
were carried out using primers previously de-
scribed (Ellis et al., 1999; Kim and Chae, 2001b).
The forward primer was 5%-CGGATATTG-
TAGTCCTGGTCG-3% (nucleotide positions
1095-1115), and the reverse primer was 5%-ACT-
GTCAAGGCTACCACAGTCA-3% (nucleotide
positions 1570-1549). The nested primers am-
plified a 225-bp fragment that was in the 481-bp
region amplified in the first reaction. The forward
primer was 5%-GATTGTATGGCGGGAG-
GAGT-3% (nucleotide positions 1286-1305), and
the reverse primer was 5%-ATTGACGACTTT-
GTTCCCCC-3% (nucleotide positions 1510-1491).
2.4. Polymerase chain reaction
Supernatant (10 ml) containing extracted DNA
was used as PCR templates in the first reaction,
and 10 ml of the product was used for the second
27
reaction. The amplification was performed in a
50-ml reaction mixture containing 1.25 mM
MgCl2, 1× PCR buffer, 0.2 mM of each dNTP, 1
mM of each primer, and 2.5 U of Taq DNA
polymerase (Perkin Elmer, Foster City, CA,
USA). Both reactions were run in a thermocycler
under (GeneAmp PCR System 9600, Perkin
Elmer-Cetus, Norwalk, CT, USA) the same con-
ditions, 35 cycles of denaturation at 95 °C for 1
min, primer annealing at 65 °C for 1 min, and
extension at 72 °C for 1 min. The PCR was ended
with a final extension step at 72 °C for 10 min.
The amplified product was visualized by stan-
dard gel electrophoresis of 10 ml of the final
reaction mixture on a 2% agarose gel. Amplified
DNA fragments of specific sizes were located by
ultraviolet fluorescence after staining with ethid-
ium bromide. Their lengths were verified by a
digested lambda DNA standard run simulta-
neously. The PCR reactions were performed in
triplicates. Control DNA from the reference
strain was included in each reaction.
2.5. Sensiti6ity and specificity assays
Sensitivities of PCV-1 and PCV-2 detections
were also estimated through serial dilutions of
DNA extracts prepared from semen. In the spe-
cificity studies, porcine reproductive and respira-
tory syndrome virus, porcine parvovirus, porcine
epidemic diarrhea virus, transmissible gastroen-
teritis, rotavirus, and classical swine fever virus
were tested independently with both sets of
primers for PCV-1 and PCV-2.
3. Results
3.1. Optimization of PCR conditions
To improve the specificity and sensitivity of the
multiplex conventional and mutiplex nested PCR
reactions, various MgCl2 concentrations (1–2
mM), primer concentrations (0.4–2 mM), anneal-
ing (50–68 °C) and denaturing (94, 95, or 98 °C)
temperatures, and cycle numbers (30, 35, 40 or
45) were tested. The optimum product yield was
achieved with 1.25 mM MgCl2, 1 mM primer,
28 J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31
Fig. 1. Multiplex conventional and nested PCR sensitivity for
PCV-1. Ten-fold dilutions of the extracted PCV-1 DNA from
10 − 3 to 10 − 5 were used. Lane M =100 bp DNA ladder; lane
1=conventional PCR for 10 − 3 dilution; lane 2 =conven-
tional PCR for 10 − 4 dilution; lane 3 =conventional PCR for
10 − 5 dilution; lane 4 = nested PCR for 10 − 3 dilution; lane
5= nested PCR for 10 − 4 dilution; lane 6 = nested PCR for
10 − 5 dilution.
annealing and denaturing temperatures of 65 and
95 °C, respectively, and 35 cycles.
3.2. Sensiti6ity and specificity assays
The multiplex conventional PCR test detected
PCV-1 DNA up to a dilution of 10 − 3, corre-
sponding to 1.9×10 − 3 TCID50 ml − 1 and PCV-2
DNA up to a dilution of 10 − 3, corresponding to
2.2 × 10 − 3 TCID50 ml − 1. The multiplex nested
PCR test, on the other hand, detected PCV-1
DNA up to a dilution of 10 − 5, corresponding to
2.3 × 10 − 4 TCID50 ml − 1 and PCV-2 DNA up to
a 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 of
the viruses tested in this study.
Fig. 2. Multiplex conventional and nested PCR sensitivity for
PCV-2. Ten-fold dilutions of the extracted PCV-2 DNA from
10 − 3 to 10 − 5 were used. Lane M =100 bp DNA ladder; lane
1=conventional PCR for 10 − 3 dilution; lane 2 =conven-
tional PCR for 10 − 4 dilution; lane 3 =conventional PCR for
10 − 5 dilution; lane 4 = nested PCR for 10 − 3 dilution; lane
5= nested PCR for 10 − 4 dilution; lane 6 = nested PCR for
10 − 5 dilution.
Fig. 3. Detection of PCV from seminal fluid fraction by
multiplex conventional PCR and multiplex nested PCR. Lane
M =100 bp DNA ladder; lane 1 =positive multiplex conven-
tional PCR for PCV-1; lane 2 = positive multiplex nested PCR
for PCV-1; lane 3 =positive multiplex conventional PCR for
PCV-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 from
sample; lane 7 = positive multiplex nested PCR for PCV-1 and
PCV-2 from sample.
3.3. Comparison of multiplex con6entional PCR
and multiplex nested PCR
A total of 18 (30%) and 30 (50%) of 60 whole
semen samples were found to be positive using the
multiplex conventional PCR and multiplex nested
PCR test, respectively (Fig. 3). All 18 samples
found to be positive by multiplex conventional
PCR were also found to be positive by multiplex
nested PCR. Of the 18 samples found to be
positive using the multiplex conventional PCR,
one tested positive for PCV-1 only, three for
PCV-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-1
only, eight for PCV-2 only, and 20 for both
PCV-1 and PCV-2. PCV-1 and PCV-2 products
obtained using multiplex nested PCR were se-
quenced, and their identities confirmed as PCV-1
and PCV-2, respectively (data not shown).
3.4. Detection of PCV from seminal fractions
Of the 22 semen samples that tested positive for
PCV-1 using multiplex nested PCR, PCV-1 DNA
was detected in 22 of the seminal fluid fractions,
nine nonsperm cell fractions, and four sperm head
fractions. Of the 28 semen samples that tested
positive for PCV-2 using multiplex nested PCR,
PCV-2 DNA was detected in 28 of the seminal
fluid fractions, 11 nonsperm cell fractions, and
four sperm head fractions (Table 1).
 J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31
3.5. Virus isolation
Attempts were made to isolate PCV-1 and
PCV-2 from 60 whole semen samples. PK-15 cell
cultures were inoculated with pooled semen ho-
mogenates from the PCR-positive animals. After
two successive passages and an incubation period
of 5 days, no cytopathic effect was observed.
However, in four of these samples (6.7%), PCV-2
nucleic acid could be detected in the cytoplasm of
infected cells using in situ hybridization. In these
four samples, the presence of PCV-2 was also
detected, using multiplex conventional PCR, in
the 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. Using
this test, DNA products with the unique size
characteristic for each type of PCV were obtained,
and sequencing then confirmed them to be type-
specific PCV. Most PCV DNA was detected in
the seminal fluid and nonsperm cell fractions of
the boar semen samples tested. Although PCV
DNA was also found to be present in four of the
sperm head fractions investigated, it is possible
that these fractions may have been contaminated
with minute amounts of PCV DNA from the
seminal fluid or nonsperm cells fractions. Infected
cells from the site of virus replication may also
find their way into the semen. This might explain
why in 11 of the 60 semen samples tested, PCV
Table 1
Detection of PCV from various seminal fractions by multiplex nested PCR
Seminal fractions
Seminal fluid Nonsperm cell
PCV-1 + −
PCV-2 + −
PCV-2 + +
PCV-1, -2 + −
PCV-1, -2 + +
PCV-1, -2 + +
29
was detected in both the seminal fluid and non-
sperm cell fractions.
Serological studies undertaken in different
countries have demonstrated that both PCV-1
and 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. In
addition, the number of seropositives for PCV-2
has been found to be greater than that for PCV-1
(Magar et al., 2000), indicating that PCV-2 is the
main PCV type circulating in the swine popula-
tion. Our results would appear to confirm that
PCV DNA is found frequently in boar semen. A
high prevalence of TT virus, a virus in the same
family as PCV, in human semen has also been
reported (Inami et al., 2000; Matsubara et al.,
2000).
The presence of PCR inhibitors in semen speci-
mens is a well-known diagnostic problem that
frequently leads to false-negative results (Krieger
et al., 1991; Coombs et al., 1993). Although the
exact mechanism for this inhibition is unknown, it
may be caused by the sperm cell DNA (van
Engelenburg et al., 1993; Wiedmann et al., 1993).
In order to prevent this problem occurring in the
present study, a detergent was used instead of
reducing agents. Reducing agents appear to cause
chromatin decondensation of sperm DNA (Even-
son et al., 1980) which may lead to false-negative
PCR 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
Number of samples
Sperm head
− 2
− 6
− 2
− 11
− 5
+ 4
30 J. Kim et al. / Journal of Virological Methods 98 (2001) 25–31
sample (data not shown), suggesting that diluting
may allow for the detection of virus in at least
some semen samples where detection might not
otherwise have been possible. The success of this
procedure may be due to the diluting of inhibitors
present in the cell fractions.
Another problem with PCR assays is the occur-
rence of false-positive results caused by the
contamination of reaction mixtures with amplifi-
cation products of a previous run in which the
same primer set was used. However, the consis-
tently negative results obtained in this present
study from the numerous negative controls used
indicate that such false-positives were not a prob-
lem, due to the care that was taken during DNA
extraction 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 further
minimize the risk of false-positive results as a
result of contamination.
The PCR test described in this study can be
completed rapidly, within 1 working day, whereas
virus isolation requires at least 1 week. In the
present study, PCV was isolated in cell cultures
from only four of the PCR positive samples
tested. Failure to isolate PCV in semen using such
a method may have been due to the cytotoxicity
of semen samples as previously reported (Med-
veczky and Szabo, 1981; Weiblen et al., 1992). It
was determined that a minimal dilution factor of
1:20 is needed when testing semen for the presence
of virus, in order to circumvent the problem of
cellular toxicity (data not shown). Furthermore, a
distinct cytopathic effect was not observed in the
cell culture, therefore requiring further serological
identification using either indirect immunofluores-
cence or in situ hybridization, in order to finally
detect and differentiate between PCV-1 and PCV-
2. As such, the multiplex nested PCR method was
found to be a more convenient, rapid, and reliable
method for the routine differential diagnosis of
PCV in semen.
The results of this study, therefore, support the
use of PCR as an alternative method to virus
isolation in the detection of and differentiation
between PCV types in semen. A high prevalence
of PCV in pig semen was also found using the
PCR assay described in this study. This latter
finding suggests that semen may be a significant
vehicle for the transmission of PCV-2. Although
this particular PCR test cannot determine the
infectivity of PCV in porcine semen, the results of
this study do indicate that it can be used to
determine whether a particular porcine semen
sample is free of PCV.
Acknowledgements
The research was supported by Ministry of
Agriculture, Forestry and Fisheries-Special
Grants Research Program (MAFF-SGRP), and
Brain Korea 21 Project, Republic of Korea.
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