Evaluation of a Nested Polymerase Chain Reaction Assay to Differentiate Between Two Genotypes of Porcine Circovirus-2

Abstract

Porcine circovirus-2 (PCV-2) is associated with several diseases in pigs, including postweaning multisystemic wasting syndrome (PMWS). A new genotype of PCV-2 was isolated from swine farms with and without clinical PMWS in North America. The new genotype was differentiated in a separate cluster by phylogenetic analyses and is now named PCV-2b compared with PCV-2a for the previously known genotype. The purpose of this study was to develop and evaluate a nested polymerase chain reaction (nPCR) assay to detect and differentiate between PCV-2a and PCV-2b. Genotype-specific primer sets were designed by using sequence data published for different PCV-2 strains. Specificity and sensitivity of the nPCR were examined by using PCV-2 isolates with known genotype. Nested PCR was found to be highly specific and sensitive for detecting and differentiating between the PCV-2 genotypes compared with the conventional 1-step PCR assay. Nested PCR was applied to detect PCV-2 and to identify the genotype in serum samples from swine farms with and without a clinical history of PMWS. Of 60 serum samples collected from 4 farms during clinical PMWS outbreaks, PCV-2a and PCV-2b were detected in 6 and 49 samples, respectively. Six of the 10 samples from one of the 4 farms had both PCV-2a and PCV-2b. Of 20 serum samples from 2 farms without PMWS, 11 were positive for PCV-2a only. These results suggest that the differential nPCR can be used to detect PCV-2 and to differentiate the 2 genotypes from field samples.

Keywords

Genotype differentiation nested polymerase chain reaction

Introduction

Porcine circovirus-2 (PCV-2; family Circoviridae, genus Circovirus) is a very small, circular, single-stranded DNA virus. Two open reading frames (ORF) of PCV-2, ORF1 and ORF2, each encode a replication protein (Rep) and a structural protein (Cap). 13 The virus has been associated with several disease syndromes in pigs, including postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome, reproductive failure, and porcine respiratory disease complex. 2,7,15 These syndromes together are considered Porcine circovirus–associated disease (PCVAD). 1,8

Isolation of a new genotype of PCV-2 was reported from pig farms with high mortality from PMWS in Canada (Gagnon CA, Tremblay D, Tijssen P, et al.: 2007, PCV2 strain variation: What does it mean? In: Proceedings of the American Association of Swine Veterinarians, pp. 535–540. AASV, Orlando, FL). 4 The new genotype was also reported in U.S. farms, both with and without clinical PMWS. 5,8 In North America, the new PCV-2 genotype is now commonly named PCV-2b to distinguish it from PCV-2a, the previously known genotype (Gagnon et al.: 2007, PCV2 strain variation). 8 In a molecular evolution study, 14 PCV-2 strains from different geographical areas were also shown to be differentiated into genetic groups 1 and 2. Genetically, PCV-2 groups 1 and 2 were found to contain 1,768 and 1,767 nucleotides, respectively. 5,14 In phylogenetic trees, group 1 viruses were classified in the same cluster of PCV-2b and were mostly reported from European countries and China, whereas group 2 viruses were from countries throughout the world. All of the PCV-2 reported from North America before 2004 were categorized in the PCV-2a group (Gagnon et al.: 2007, PCV2 strain variation). 5 This coincided with the initial experience with high mortality from PMWS in swine farms in Canada and the United States (Gagnon et al.: 2007, PCV2 strain variation). 5

The polymerase chain reaction (PCR) assay has been used as a specific and sensitive diagnostic method for the detection of PCV-2 in field samples. 3,10,11 By using a PCR assay, PCV-2 can readily be differentiated from Porcine circovirus-1 (PCV-1). Conventional 1-step PCR is sensitive enough to detect PCV-2 in tissue samples; however, it is not sensitive enough for use with serum and semen samples, because of an insufficient quantity of the viral DNA. Therefore, nested PCR (nPCR) assays were developed and applied to detect PCV-2 for those samples. 9,12

Distinguishing between PCV-2a and PCV-2b is difficult and time consuming. PCV-2 genotypes have been classified by several methods, including viral genomic sequencing, restriction fragment length polymorphism (RFLP) analysis, real-time PCR assay, and genotype-specific PCR (Gagnon et al.: 2007, PCV2 strain variation). 4,8 Each of these methods has advantages and disadvantages in obtaining the results. It is generally accepted that nPCR is less expensive than real-time PCR, and less time consuming and labor consuming than RFLP and sequencing methods.

It has been understood that the genotype-specific primers for differential PCR with high sensitivity are not easy to design, because the genetic identity between the 2 genotypes is very high (>95%). 14 The differentiation between PCV-2a and PCV-2b by using a nPCR assay has not been reported, and the nPCR reported previously 12 was used solely to detect PCV-2 without genotype differentiation from samples with low quantities of virus, such as semen. Therefore, the purpose of the current study was to develop and evaluate a nPCR assay to detect and differentiate between the 2 genotypes PCV-2a and PCV-2b. The nPCR with specific primer sets for each group was evaluated for specificity and sensitivity by using PCV-2 of a known genotype. The nPCR was also applied on field serum samples for the detection and genotyping of PCV-2.

Materials and methods

Minnesota PCV-2 isolates, sequencing, and phylogenic analysis

Eight PCV-2 isolates and 6 uncultured PCV-2 PCR-positive products from Minnesota farms with or without PMWS were used for phylogenetic analysis and a specificity test of nPCR. The ORF2 sequences of the 14 PCV-2 samples were analyzed in comparison with 22 ORF2 gene sequences of PCV-2 strains in GenBank from different countries in North America, Europe, and Asia. For the sequencing, PCR products were purified by using a QIAEXII commercial gel purification kit a according to the manufacturer's instructions. Purified PCR products were sequenced by using the electrophoretic DNA analyzers, ABI 3730xl. b Nucleotide sequence alignment and phylogenic analysis were performed by Neighbor-Joining tree by using the ClustalX version 1.83 alignment program. c Bootstrap values were evaluated on 500 repeats of the alignment.

Primer design for nPCR

Primer sets that could detect the 2 genotypes of PCV-2 were designed. Nucleotide sequence data for PCV-2 strains from GenBank and the 14 Minnesota PCV-2 isolates were aligned by using ClustalX 1.83 to identify regions that differed between the 2 genotypes. The most heterogeneous nucleotide portion of the 2 genotypes was detected from 1462–1480, and reverse primers of 2 types were designed (Fig. 1). To amplify the PCR products of different sizes, the forward primer for PCV-2a genotype was designed in both ORF1 and ORF2 regions, and the primer of PCV-2b was in the ORF2 region. The primer sequences for PCV-2a were 2a-F (forward): 5′-AACAATCCACGGAGGAAGG-3′ and 2a-R (reverse): 5′-GGGACCAACAAAATCTCY-3′, and for PCV-2b were 2b-F (forward): 5′-CTGTTTTCGAA CGCAGTG-3′ and 2b-R (reverse): 5′-CTCAAACCCCC GCTCTG-3′. The predicted product sizes of the PCV-2a–specific and PCV-2b–specific PCR were 360 base pair (bp) and 568 bp, respectively. For the first-round PCR, the reverse nested primer was designed from the highly conserved region outside 2a-R primer sites in ORF2. The forward nested primer, which can differentiate PCV-1 and PCV-2, was selected according to the results from a previously study. 11 The primer sequences were VF-2 (forward): 5′-GAAGAATGGAAGAAGCGG-3′ and nest-R (reverse): 5′-ACAGTCAGAACGCCCTCCT-3′. The predicted PCR product size of the first-round primer was 1494 bp. The oligonucleotide primer sets were designed by using the Primer3 (version 0.3.0) program. d

Figure 1.

A diagram of primer design. The reverse primers 2a-R and 2b-R commonly recognize the most distinct portion between Porcine circovirus-2 (PCV-2) genotypes 2a and 2b. The forward primers 2a-F and 2b-F were designed to make different polymerase chain reaction product sizes in open reading frames of PCV-2, open reading frames (ORF) 1 and ORF2, respectively. The forward primer of the first round, VF-2, can differentiate Porcine circovirus-1 (PCV-1) and PCV-2 in which a few nucleotides are deleted in PCV-1.

PCR and nPCR assays

Genomic viral DNA from PCV-2 isolate and serum sample was extracted and purified by using DNeasy Blood and Tissue kit a as described in the manufacturer's manual. The first-round PCR was performed with the VF-2 and nest-R primer set. A final volume of 25 μl contained 2 μl of extracted viral DNA, 2.5 μl 5X reaction buffer (200 mM of Tris-HCl, pH 8.4, 500 mM of KCl), 2.0 mM of MgCl₂, 1 μl of 1 pM each primer, 0.25 mM of dNTPs (deoxyribonucleotide triphosphates), and 1 U of Taq polymerase, e and adjusted with DNase-free distilled water to make the final volume. The amplification was undertaken by using a thermal cycler f and the following cycling profile: 5 min denaturation at 94°C, 30 repetitive cycles of 30 sec of denaturation at 94°C, 30 sec of annealing at 54°C, and 40 sec of elongation at 72°C. Elongation was extended for 7 additional min in the last cycle.

For differential nPCR, the amplified product of the first-round PCR was reacted with each group specific primer set in separate tubes. The primary amplification product of 1 μl was added to a new PCR mixture of 5X reaction buffer (200 mM of Tris-HCl, pH 8.4, 500 mM of KCl), 2.0 mM of MgCl₂, 1 μl of 3 pM each primer, 0.25 mM of dNTP, and 1 U of Taq polymerase, e and adjusted with DNase-free distilled water to make 25 μl of the final volume. The nPCR steps for both group-specific reactions were performed as described above for PCR, except for 30 sec of annealing at 57°C and 30 sec of elongation at 72°C. The amplified PCR products were analyzed by 1.5% agarose gel electrophoresis and examined under ultraviolet light.

Specificity and sensitivity of the nPCR

Initially, nPCR was used to evaluate specificity by using 2 reference PCV-2 isolates of known genotype: EF452353 (PCV-2a) and EF452350 (PCV-2b). With the reference strains, the viral DNA was reacted with first-round primer set and then the PCR products were performed with 2 differential primer sets. In a tube, the first-round PCR product of EF452353 (PCV-2a) was reacted with PCV-2a genotype-specific primer set. At the same time, the PCR product was reacted with PCV-2b genotype-specific primer set in another tube. Similarly EF452350 (PCV-2b) was also tested with PCV-2a–specific and PCV-2b–specific primer sets. Porcine circovirus-1 and Porcine parvovirus (PPV) were also tested with the same nPCR procedure for its specificity. Subsequently, all 14 Minnesota PCV-2 isolates (PCV-2a: EF452352, EF452353, EF452364, EF452365, and EF452367; PCV-2b: EF452350, EF452351, EF452354, EF452356, EF452358, EF452359, EF452360, EF452361, and EF452363) were tested by nPCR. Viral DNA from each virus was tested with both differential primer sets and examined for the presence of nonspecific primer reaction. The PCR products of 360 and 568 bp obtained in the second-round PCR during development stage were sequenced to confirm specificity.

The sensitivity of the nPCR was examined in comparison with conventional 1-step PCR. The EF452353 (PCV-2a) and EF452350 (PCV-2b) strains were preadjusted to be the same infectivity titer (10^4.0 TCID₅₀/ml) and were serially diluted 10-fold up to 10^-10. Viral DNA of each dilution was tested by both nPCR and 1-step PCR without the first-round step. The nPCR was further performed with 12 serum samples from 2 different farms in which each genotype of PCV-2 had been detected. These samples were examined by conventional 1-step and nPCR assays, and the results were compared.

Application of nPCR assay on serum samples

A total of 80 serum samples from pigs between 8 and 14 weeks old from 6 different swine farms were randomly selected from a serum bank. Farms A, B, C, and D had a history of typical clinical signs of PMWS during the time of blood collection in 2006. Sera collected in 2004 from farms E and F were also used. Farms E and F had no history of clinical PMWS, although PCV-2 infection was diagnosed. Viral DNA extraction and nPCR were carried out to detect and differentiate PCV-2 genotypes as described above.

Figure 2.

Phylogenetic tree based on the open reading frame 2 gene of Porcine circovirus-2 (PCV-2) Minnesota isolates. Fourteen PCV-2 isolates from Minnesota and 22 PCV-2 strains from other geographic regions were compared. GenBank accession number and geographic region for each isolate are shown. CAN = Canada; FRA = France; UK = United Kingdom; NETH = Netherlands; US = United States; MN = Minnesota.

Results

Phylogenic analysis of Minnesota PCV-2 isolates

Two distinct genetic clusters were identified when ORF2 genes of the 14 PCV-2 Minnesota isolates and 22 previously published PCV-2 strains from GenBank were phylogenetically analyzed (Fig. 2). Five Minnesota isolates in the upper cluster were categorized as PCV-2a together with 13 North American PCV-2 strains that were reported before 2001. The lower cluster classified as PCV-2b was composed of 9 Minnesota isolates and 1 U.S. strain reported in 2006, together with other European and Chinese strains. The 5 PCV-2a and the 9 PCV-2b isolates were from samples that originated at 3 farms without PMWS and 5 farms with PMWS, respectively.

Specificity of nPCR

The primer pairs designed to differentiate between 2 genotypes of PCV-2 specifically amplified each PCV-2 genotype without any nonspecific reaction. As shown in Figure 3a, viral DNA of PCV-2b (EF452350) yielded a positive reaction with the 2b-F and 2b-R primer set but not with the PCV-2a primer set. Porcine circovirus-2, genotype 2a (EF452353) was amplified only with the 2a-F and 2a-R primer set. Porcine circovirus-1 and PPV were not detected by any primer set, and neither the first-round nor the second-round primer sets cross-reacted with any PCR reaction (Fig. 3a). Four Minnesota PCV-2a isolates (EF452352, EF452364, EF452365, and EF452367) were detected by the PCV-2a–specific nPCR assay, and 8 Minnesota PCV-2b isolates (EF452351, EF452354, EF452356, EF452358, EF452359, EF452360, EF452361, and EF452363) were detected by the PCV-2b–specific nPCR (Fig. 3b). There was no known nonspecific PCR product with these PCV-2 isolates by cross-reaction of the primer sets (data not shown).

Figure 3.

Specificity of nested polymerase chain reaction. a, M: 100–base pair (bp) marker; lane 1: Porcine circovirus-2 (PCV-2) genotype 2b (EF452350) by PCV-2b–specific primer set; lane 2: PCV-2b (EF452350) by PCV-2a–specific primer set; lane 3: PCV-2a (EF452353) by PCV-2b–specific primer set; lane 4: PCV-2a (EF452353) by PCV-2a–specific primer set; lanes 5, 6: Porcine circovirus-1 (PCV-1); lanes 7, 8: Porcine parvovirus; lanes 9, 10: uninfected porcine kidney (PK)-15 cells; lanes 11, 12: distilled water by PCV-2a– and PCV-2b–specific primer sets. b, Lanes 1–10: 8 PCV-2b isolates of EF452351, EF452354, EF452356, EF452357, EF452359, EF452360, EF452361, EF452363, uninfected PK-15 cells, and distilled water by PCV-2b–specific primer set; lanes 11–16: 4 PCV-2a isolates of EF452352, EF452364, EF452365, EF452367, uninfected PK-15 cells, and distilled water by PCV-2a–specific primer set.

Sensitivity of nPCR

The analytical sensitivity of the nPCR was examined by comparing the results by 1-step and nPCR assays by using viral DNA extracts from 10-fold serially diluted samples of the reference PCV-2a and PCV-2b. Both genotypes of PCV-2 with an infectivity titer of 10^4.0 TCID₅₀/ml were detected up to 10^-5 dilution by 1-step PCR, whereas the viruses were detected up to 10^-9 dilution by nPCR (Fig. 4). When the nPCR was performed on 12 sera from 2 different farms with a known clinical history, PCV-2a was detected in 5 sera from a farm with no history of PMWS, whereas PCV-2b was detected in 7 sera from another farm with clinical PMWS. However, in the 1-step PCR, only 5 of the 12 sera were positive (Fig. 5). One positive and 1 weak-positive band were detected with the 5 PCV-2a–positive sera, and 1 positive and 2 weak-positive bands were detected with 7 PCV-2b–positive sera. All of the 12 PCR-positive samples were sequenced, and their genotypes were confirmed.

Figure 4.

Detection of Porcine circovirus-2 (PCV-2) genotypes 2a and 2b in different dilutions. Both PCV-2a (EF452353) and PCV-2b (EF452350) were preadjusted to be 10^4.0 TCID₅₀/ml. a, One-step polymerase chain reaction (PCR); left 4 lanes: 10^-3–10 6 dilution of PCV-2b (EF452350); right 4 lanes: 10^-3–10^-6 dilution of PCV-2a (EF452353). b, Nested PCR; left 6 lanes: 10^-5–10^-10 dilution of PCV-2b (EF452350); right 6 lanes: 10^-5–10^-10 dilution of PCV-2a (EF452353). Lanes C and W in panels (a) and (b): uninfected porcine kidney (PK)-15 cells and distilled water by PCV-2a– and PCV-2b–specific primer sets.

Examination of field serum samples

The detection of PCV-2 genotypes for 80 serum samples from 6 different swine farms are summarized in Table 1. Of the 60 samples from farms A, B, C, and D with clinical PMWS, PCV-2b was detected from 49 samples (81.7%). Six samples from farm C were positive for both PCV-2a and PCV-2b. Of the 20 serum samples from farms D and E without PMWS, 11 samples were positive for PCV-2a, and none were positive for PCV-2b.

Discussion

Porcine circovirus-associated disease is now considered a global disease and the cause of significant economic losses in the swine industry. In a case of PCVAD at the University of Minnesota research farm during mid 2005, 48 of 340 grower pigs (14.1%) died or were culled because of severe clinical signs of PMWS. Porcine circovirus-2 was isolated from the samples of pigs with PMWS signs. Since that time, samples from many farms both with and without PMWS were tested to isolate PCV-2 and to examine their genetic characteristics. By using phylogenetic analysis, genetic similarity was observed between the Minnesota isolates and various European strains. As shown in Figure 2, the PCV-2 isolates and PCR-positive samples were classified into 2 genetic clusters. All of the 9 Minnesota PCV-2 isolates in the lower cluster were from pigs with clinical PMWS. This finding led to an investigation of an association between the isolation of PCV-2b and the presence of clinical PMWS on farms. The first step for the investigation was to develop a sensitive, specific, and differential PCR assay for the 2 genotypes.

Figure 5.

Comparison of Porcine circovirus-2 (PCV-2) detection by 1-step (a) and nested polymerase chain reaction (b) in serum samples. In panels (a) and (b), lanes 1–7: PCV-2b–positive serum samples from a farm with postweaning multisystemic wasting syndrome (PMWS); lanes 11–15: PCV-2a–positive samples from another farm without PMWS. Lanes 8 and 16 and lanes 9 and 17 are uninfected porcine kidney (PK)-15 cells and distilled water, respectively. Lanes 10 and 18 are positive controls for PCV-2b (EF452350) and PCV-2a (EF452353), respectively.

Developing a differential PCR assay between PCV-1 and PCV-2 would be relatively easy compared with developing an assay between PCV-2a and PCV-2b. Porcine circovirus-1 and PCV-2 have higher genetic heterogeneity. For differential PCR between PCV-2a and PCV-2b, the main problem was that the choices of optimal positions for the primers were highly limited. Also, the designed primer set was able to amplify various nonspecific products because of the high level of genetic identity, even with modifications in PCR conditions. Initially, nested differential PCR was performed in 1 tube, but the PCR reaction showed more than 2 product bands. The additional nonspecific band was believed to be because of cross-reaction of the primers between 2 types of viral DNA. Therefore, the subsequent assays were carried out in 2 separate PCR tubes, each containing a specific primer set with 2 groups of viral DNA.

To develop a nPCR in the current study, a primer set for the first-round PCR was designed on the outside positions of each group-specific primer, and the designed primer sequences were further modified after comparing optimal PCR conditions. However, although the sensitivity of PCR was improved, nonspecific PCR products were amplified by both PCV-2a–specific and PCV-2b–specific primer sets in PCV-1 viral DNA, which had not been previously detected by conventional differential PCR. Therefore, the forward primer of first-round PCR was replaced with another primer that can differentiate PCV-1 and PCV-2, as found in a previous study. 11

The nPCR, compared with conventional PCR, was more sensitive in the detection of PCV-2 from serum samples as described by others. 9,12 The serum samples from PCV-2–infected pigs were often negative by a conventional PCR assay. The quantity of viral DNA in the sera was lower than in lungs or lymph nodes by up to 1,000 times. 6 The nPCR assay in the present study was highly sensitive and detected up to 10 4 times more than conventional PCR. In the current study, conventional PCR detected PCV-2 in only 5 of the 12 samples (42%) that tested positive by nPCR. This result agreed with a previous report that showed that PCV-2 from serum samples of 54% were detected by a conventional PCR compared with those with a nPCR. 9

One great advantage of the nPCR described in the present study is that it cannot only differentiate between the genotypes but can also detect dual infection of both genotypes (Table 1). In addition, the assay was found to be highly specific, and no false-positive band was observed with PCV-1 or PPV (Fig. 3). Two genotypes of PCV-2 were clearly differentiated by the nPCR, and there was complete agreement between the results by the nPCR and phylogenetic analyses (Fig. 2) with PCV-2 isolates from the Minnesota swine farms.

It is interesting to note that PCV-2b was not detected in the farms without clinical signs of PMWS. In the farms with clinical PMWS, PCV-2b alone or dual genotypes of PCV-2 were detected. These findings were similar to the previous reports (Gagnon et al.: 2007, PCV2 gene variation), 5,8 which observed that the sudden appearance of PCV-2b was associated with an increase in PMWS farms in North Carolina, Iowa, and Kansas, and suggested that the outbreaks of PCV-2b were a concern for the swine industry. 5 There have been debates within the scientific community on the difference in virulence between the 2 genotypes of PCV-2. Comparative experimental infection of PCV-2a and PCV-2b in susceptible pigs will be necessary to conclude any relation between the virulence and the genotype of PCV-2.

Table 1.

Differentiation between Porcine circovirus-2 (PCV-2) genotypes 2a and 2b by nested polymerase chain reaction in serum samples collected from swine farms with a known clinical history of postweaning multisystemic wasting syndrome (PMWS).

				PCV-2 genotype detected
Pig farms	Clinical PMWS	Year sample collected	No. sera tested	PCV-2a	PCV-2b	PCV-2a+2b
A	Yes	2006	30	0	23	0
B	Yes	2006	10	0	10	0
C	Yes	2006	10	6	10	6
D	Yes	2006	10	0	6	0
E	No	2004	10	5	0	0
F	No	2004	10	6	0	0

In the present study, the main goal was to develop a nPCR assay for the differentiation of the 2 genotypes of PCV-2. It is believed that nPCR combines specificity and sensitivity, and reduces the labor and time intensiveness of diagnosing PCV-2a and PCV-2b. It is interesting that, although a limited serum sample was used in this study, PCV-2b was detected in the sera from swine farms that were diagnosed with PMWS in 2006, whereas PCV-2a was mainly detected in samples from the swine farms without PMWS in 2004. This finding could potentially initiate different studies by using the differential nPCR. An epidemiologic study with a large sample size will be needed to determine the relationship between the detection of PCV-2b and the presence of clinical PMWS in swine farms. The prevalence of PCV-2 genotypes can also be compared in sera from swine farms during a PMWS outbreak and after the recovery. The differential nPCR can be used in the pathogenesis studies with pigs after inoculation of 2 different genotypes of PCV-2. Finally, differential nPCR could be useful for examining semen samples in boar studs. In conclusion, nPCR promises to be a valuable tool for diagnosing PCV-2 in pigs.

Acknowledgements

The study was funded by a grant from MJ Biologics, Mankato, MN. The authors thank Dr. Karin Matchett for a critical reading of this manuscript.

Footnotes

a.

Qiagen, Inc., Valencia, CA.

b.

BioMedical Genomic Center, University of Minnesota, St. Paul, MN.

c.

Plate-Forme de Bio-informatique, Illkirch, France.

d.

Source Forge, Inc., Mountain View, CA ().

e.

Promega Corp., Madison, WI.

f.

PerkinElmer Life and Analytical Sciences, Inc., Waltham, MS.

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