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1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2 Department of Health Care and Biotechnology, KATHO Catholic University College of South-West Flanders, Wilgenstraat 32, 8800 Roeselare, Belgium
Correspondence
H. J. Nauwynck
hans.nauwynck{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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91 %), antigenic differences at the capsid protein level are present among PCV-2 strains with a different genetic and clinical background. The GenBank/EMBL/DDBJ accession numbers of the ORF2 sequences from strains 1206, VC2002-k2 and VC2002-k39 determined in this study are EF990644, EF990645 and EF990646, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1982). PCV-1 is not regarded as a pathogen for pigs (Tischer et al., 1986; Allan et al., 1995), whereas PCV-2 is considered as the crucial pathogen in post-weaning multisystemic wasting syndrome (PMWS), a multifactorial swine disease that causes wasting and death in weaned piglets (Harding, 1996; Nayar et al., 1997; Ellis et al., 1998; Allan & Ellis, 2000). Besides wasting, PCV-2 may also cause reproductive failure (West et al., 1999; Ladekjær-Mikkelsen et al., 2001; Meehan et al., 2001; Sanchez et al., 2001, 2003; Mateusen et al., 2004). PCV-2 has also been isolated from pigs with porcine dermatitis and nephropathy syndrome (PDNS) and a number of other diseases, but neither PDNS nor these other diseases have been reproduced experimentally (Allan et al., 2000; Rosell et al., 2000; Segales et al., 2005, Wellenberg et al., 2004).
The PCV-2 virion measures approximately 17 nm in diameter, is non-enveloped and consists of a circular single-stranded DNA surrounded by an icosahedral capsid (Allan et al., 1998). The ambisense DNA molecule contains about 1.77 kb and 11 putative open reading frames (ORFs) (Hamel et al., 1998). Proteins encoded by three of these ORFs are considered to play a role in the pathogenesis of PCV-2 infections. ORF1 encodes the replication-associated proteins Rep and Rep' (Mankertz & Hillenbrand, 2001; Cheung, 2003; Mankertz et al., 2003). Rep and Rep' are 37.5 and 20.2 kDa, respectively. ORF2 encodes the 27.8 kDa capsid protein (Hamel et al., 1998; Meehan et al., 1998; Morozov et al., 1998; Mankertz et al., 2000; Nawagitgul et al., 2000). The ORF2 protein is the only structural protein. The ORF3 protein has a molecular mass of 11.8 kDa and has recently been associated with apoptosis in vitro and with viral pathogenesis in mice (Liu et al., 2005, 2006).
Meerts et al. (2005a) demonstrated biological differences among different PCV-2 strains in vitro. Replication kinetics of PMWS- and PDNS-associated PCV-2 strains were significantly different from reproductive failure-associated PCV-2 strains. Recently, it was demonstrated that the virulence of a PCV-2 isolate originating from a PMWS-affected animal differed significantly from an isolate recovered from a subclinically infected animal. Important differences in serological profile, virus replication and severity of lesions were shown after experimental inoculation of specific-pathogen-free (SPF) pigs (Opriessnig et al., 2006).
Among various strains of PCV-2, the identity at the nucleotide level of the Rep protein and the capsid protein is 97–100 % and 91–100 %, respectively. At protein level, identity is 97–100 % for Rep and 89–100 % for the capsid protein (Larochelle et al., 2002). Several studies have suggested that genetic differences in PCV-2 are associated with the geographical region from which the isolates originated (Fenaux et al., 2000; Hamel et al., 2000; Mankertz et al., 2000; Kim & Lyoo, 2002) and a recently proposed classification system (Olvera et al., 2007) divides PCV-2 into two genotypes (1 and 2) and eight clusters (1A–1C and 2A–2E). Although several antigenic domains have been discovered on the capsid protein (Mahé et al., 2000; Lekcharoensuk et al., 2004; Olvera et al., 2007), no association has been established so far between the sequence of the capsid and the pathogenicity of a PCV-2 strain (Fenaux et al., 2000; Meehan et al., 2001; Larochelle et al., 2002; Pogranichniy et al., 2002; de Boisséson et al., 2004; Grierson et al., 2004). Until now, mouse monoclonal antibodies (mAbs) directed against PCV-2 have not shown major differences in reactivity to different PCV-2 strains (Allan et al., 1999; McNeilly et al., 2001).
In this study, mAbs to PCV-2 were produced, characterized and used to identify antigenic differences among PCV-2 strains with a different genotype and originating from different clinical presentations.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Olvera et al., 2007) are shown in Table 1. The replication kinetics of these strains have been documented previously (Meerts et al., 2005a). PCV-1 originated from the persistently infected PK-15 cell line ATCC CCL-33 (Tischer et al., 1974, 1982). All PCV-2 strains used were passaged 11–17 times, except for strain 1121, which was passaged 30 times.
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Cells.
PCV-negative PK-15 cells and the persistently PCV-1-infected PK-15 cell line ATCC CCL-33 were grown in minimal essential medium (MEM) containing Earle's salts (Gibco), supplemented with 5 or 10 % fetal bovine serum (FBS), 0.3 mg glutamine ml–1, 100 U penicillin ml–1, 0.1 mg streptomycin ml–1 and 0.1 mg kanamycin ml–1. Cell cultures were maintained at 37 °C in the presence of 5 % CO2.
Mouse immunization.
Before immunization, mice were made immunotolerant to PK-15 cells as described by Matthew & Sandrock (1987). Four 6-week-old female BALB/c mice were injected intraperitoneally (i.p.) with 1.5x107 PCV-negative PK-15 cells in 300 µl PBS. After 10 min, 24 and 48 h, cyclophosphamide (Sigma) was injected i.p. at a dose of 100 mg (kg body weight)–1 in a total volume of 500 µl PBS. After 3 and 6 weeks, injections with PK-15 cells and cyclophosphamide were repeated. Two weeks after the last treatment, 2.25x107 Stoon-1010-inoculated PK-15 cells were injected i.p. in a volume of 300 µl PBS mixed with an equal amount of complete Freund's adjuvant (Sigma). At this time point and 2 weeks later, serum was collected from mice. Three weeks after the inoculation with PCV-2, one mouse received an i.p. injection with 4.5x107 Stoon-1010-inoculated PK-15 cells diluted in 600 µl PBS. Euthanasia was performed 4 days later and the spleen was collected.
Production and screening of hybridomas.
Hybridoma cells were produced by fusion of spleen cells with SP 2/0 myeloma cells as described by Galfre & Milstein (1981). The resulting hybridoma cells were maintained in RPMI 1640 (Gibco) supplemented with 10 % FBS. PCV-2-specific mAbs in supernatant fluids were demonstrated on PCV-negative and Stoon-1010-inoculated PK-15 cells by an immunoperoxidase monolayer assay (IPMA) adapted from Labarque et al. (2000). After incubation with undiluted supernatant fluids for 1 h at 37 °C, cells were washed twice with PBS. Subsequently, a 1 : 500 dilution of horseradish peroxidase-labelled goat anti-mouse polyclonal antibodies (pAbs) (Dako) in PBS was added for 1 h at 37 °C. After washing twice in PBS, substrate solution was added and cell cultures were analysed by light microscopy (Olympus Optical Co.). Selected hybridoma cultures were cloned by limiting dilution.
Determination of mAb class.
The isotype of the produced mAbs was determined using a peroxidase-based commercial mouse mAb identification kit (Zymed). This test identifies the IgG1, IgG2a, IgG2b, IgG3, IgA and IgM isotype classes and the 
and 
type of the light chain using monospecific rabbit pAbs. Supernatant fluids of anti-PRV mAbs 13D12 (IgG1) and 1C11 (IgG2a) (Nauwynck & Pensaert, 1995) and anti-Escherichia coli mAb E7G3 (IgG3) (Tiels et al., 2007) were used as positive controls.
Indirect immunofluorescence staining of recombinant PCV-2 VLPs.
The VLP staining technique was adapted from Misinzo et al. (2005). Briefly, purified VLPs were diluted 1 : 100 in PBS, smeared onto microscope slides, air dried and fixed with 3 % (w/v) paraformaldehyde in PBS for 10 min at room temperature. Fixed VLPs were incubated with undiluted hybridoma supernatants for 1 h at 37 °C, followed by incubation with a 1 : 500 dilution of fluorescein isothiocyanate-labelled goat anti-mouse pAbs (Molecular Probes) containing 10 % PCV-2-negative goat serum for 1 h at 37 °C. mAb F217 (McNeilly et al., 2001) diluted 1 : 50 in PBS was used as a positive control. mAbs 13D12 and 1C11 were included as negative controls. A Leica DM/RBE fluorescence microscope (Leica Microsystems) was used for visualization.
Western blot analysis.
Stoon-1010-inoculated and mock-inoculated PCV-negative PK-15 cells were harvested by scraping. Cells were pelleted by centrifugation at 15 700 g for 20 min at 4 °C and subsequently lysed for 1 h at 37 °C in TNE [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA] containing 1 % NP-40 (Roche), protease inhibitors (Complete; Roche) and 0.5 % SDS. Cells were centrifuged at 15 700 g for 10 min at 4 °C and resuspended in a non-reducing Laemmli buffer. This mixture was boiled for 5 min and stored at –20 °C until use. Proteins were separated by standard SDS-PAGE (Laemmli, 1970) and transferred to a PVDF membrane (Amersham Biosciences). This membrane was then incubated for 1 h at room temperature in PBS containing 0.1 % Tween 20 (PBS-Tween), supplemented with 5 % BSA (Sigma). After washing in PBS-Tween, membranes were incubated overnight at 4 °C with a 1 : 5 dilution of the mAbs in PBS-Tween. mAb F190 (McNeilly et al., 2001) and biotinylated purified porcine pAbs, originating from a PCV-2-negative SPF pig inoculated with strain 1121 (Pensaert et al., 2004; Meerts et al., 2005a), were used as positive controls. mAbs 13D12 and 1C11 were included as negative controls. Next, a 1 : 300 dilution of biotinylated sheep anti-mouse pAbs and a 1 : 300 solution of a streptavidin–biotinylated horseradish peroxidase complex (Amersham Biosciences) was applied. Membranes were washed twice with PBS-Tween between incubations. Antigen–antibody complexes were visualized using an enhanced chemiluminescence assay (Amersham Biosciences).
Reactivity of mAbs to different PCV-2 strains.
PCV-2 strains Stoon-1010, 48285, 1206, VC2002, 1147, 1121 and 1103 were used to prepare 96-well IPMA plates as described by Labarque et al. (2000). PCV-negative PK-15 cells and the persistently PCV-1-infected PK-15 cell line were used for control IPMA plates. The staining procedure was similar to the IPMA technique described above. Tenfold dilutions of hybridoma supernatants were prepared in PBS and used as primary antibodies. IPMA antibody titres of a hybridoma supernatant were expressed as the reciprocal of the last dilution that resulted in a positive reaction. These assays were performed three times for each strain.
Sensitive neutralization assays.
In order to detect the neutralizing activity of the mAbs, a sensitive neutralization assay was adapted from the method of Meerts et al. (2005b). Briefly, 104.3 TCID50 PCV-2 in a volume of 200 µl was incubated for 1 h at 37 °C with 200 µl undiluted hybridoma supernatant. After incubation, this mixture was added to semi-confluent monolayers of PCV-negative PK-15 cells in four wells of a 96-well plate. After 1 h at 37 °C, cell cultures were washed twice in MEM and fresh medium was added. Cell cultures were fixed 36 h later. At this time point, the first replication cycle of PCV-2 was completed (Meerts et al., 2005a). PCV-2-infected PK-15 cells were stained by an IPMA using porcine PCV-2-specific pAbs, originating from a Stoon-1010-inoculated gnotobiotic pig. The number of infected cells per well was determined by light microscopy. The neutralizing activity of a hybridoma supernatant was expressed as the percentage reduction in the number of infected cells in comparison with medium. Assays were performed with all seven strains. Anti-PCV-2 mAb F190 was used as a positive control. mAbs 13D12 and 1C11 were used as negative controls. A mAb was considered as neutralizing when its mean neutralizing activity was higher than the mean neutralizing activity+SD of the negative controls. Sensitive neutralization experiments were performed three times for each strain.
Sequencing of ORF2 from strains 1206 and VC2002.
The Belgian PCV-2 strains 1206 and VC2002 were purified by ultracentrifugation at 180 000 g for 3 h through a 30 % sucrose gradient as described by Delputte et al. (2002). A set of PCR primers was designed based on alignment of the genome sequences of strains Stoon-1010, 48285, 1147, 1121 and 1103. The primer set PCV-2-FW (5'-AGCGCACTTCTTTCGTTTTCAG-3') and PCV-2-REV (5'-GAATGCGGCCGCTTATCACTTCGTAATGGTTTTTATTATTCA-3') amplified the complete ORF2. Two internal oligonucleotides were synthesized: CV1 (5'-GGGCTGTGGCCTTTGKTAC-3') and CV2 (5'-TGTRGACCACGTAGGCCTCG-3'). These internal oligonucleotides were as described by Fenaux et al. (2000) with minor modifications, and were used for sequencing. A 1 : 200 fraction of proteinase K-treated ultrapurified PCV-2 was used as template in PCRs using Platinum Pfx DNA polymerase (Invitrogen) at an annealing temperature of 60 °C and using the cycling conditions described by the manufacturer. PCR products (
800 bp) were treated with exonuclease I and antarctic phosphatase (New England Biolabs) and used directly for cycle sequencing with a Big Dye Terminator Cycle Sequencing kit v1.1 (Applied Biosystems) and PCV-2 primers. Cycle sequencing reaction products were purified using ethanol precipitation and separated on an ABI Genetic Analyzer 310 (Applied Biosystems). Additionally, PCR products (800 bp) were gel purified using a QIAquick gel extraction kit (Qiagen) and cloned in pBluescript II SK(+) cut with EcoRV and treated with antarctic phosphatase. Clones containing the PCV-2 ORF2 were sequenced using T7 and T3 primers as described above. The sequences were analysed and compiled using Align, LAlign, CLUSTAL W and Sixframe in the Biology Workbench (http://workbench.sdsc.edu) and Align2sequences, BLASTN and BLASTP at www.ncbi.nlm.nih.gov. Phylogenetic relationships among sequences were analysed as described by Tripathi & Sowdhamini (2006). Briefly, phylogenetic trees were derived from multiple sequence alignments with PHYLIP v3.67. Bootstrapping was performed 100 times using SEQBOOT. Pairwise distances between genomic sequences and protein sequences were determined with DNADIST and PROTDIST, respectively. Neighbour-joining (NJ) trees were calculated with neighbour and maximum-likelihood (ML) trees with DNAML and ProML. Majority rule consensus trees were obtained with CONSENSE and visualized with DRAWGRAM.
The ORF2 sequences (702 nt, from ATG to the stop codon) from strains 1206, VC2002-k2 and VC2002-k39 have been deposited in GenBank under accession numbers EF990644 [GenBank] , EF990645 [GenBank] and EF990646 [GenBank] , respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Production and screening of hybridomas
Forty-four hybridomas that produced mAbs against PCV-2-infected PK-15 cells were frozen. Cloning by limiting dilution resulted in 16 stable hybridomas that produced mAbs with an IPMA titre of 1000 or more to Stoon-1010.
Determination of mAb class
A commercial identification kit was used to determine the isotypes of the mAbs. The results are presented in Table 2. Six hybridomas produced IgG1 mAbs and eight hybridomas produced IgG2a mAbs. mAb 21C12 had an IgG3 isotype. The isotype of mAb 48B5 could not be determined. All mAbs, including mAb 48B5, had a light chain of the type.
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Western blot analysis
The reactivity of the mAbs to Stoon-1010-inoculated PK-15 cells was determined in a Western blot assay. mAbs 31D5, 38C1 and 108E8 gave a strong and specific reaction with a protein of approximately 28 kDa (Fig. 1). For mAb 21C12, a faint but specific band was observed at 28 kDa (not shown). None of the other mAbs showed reactivity in the Western blot assay.
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). Eleven out of 16 hybridomas stained all seven strains with a maximum tenfold variation in titres among the strains (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8). mAbs 31D5, 48B5, 59C6 and 108E8 did not react with the genotype 1 strains 48285, VC2002 and 1147, or they had IPMA antibody titres to these strains that were at least 100 times lower than for the genotype 2 strains Stoon-1010, 1121 and 1103. These four mAbs stained two different populations of infected cells in strain 1206. IPMA antibody titres for the first population (99 % of the infected cells) were comparable to those of the other genotype 1 strains. IPMA antibody titres for the second population (1 % of the infected cells) were comparable to those of the genotype 2 strains. These populations were determined by counting the number of infected cells per well after staining with different dilutions of the mAbs. mAb 13H4 stained all four PMWS-associated strains (Stoon-1010, 48285, 1206 and VC2002) and the single PDNS-associated strain (1147), but did not react with the two reproductive failure-associated strains (1121 and 1103). None of the 16 mAbs reacted with PCV-1 or with PK-15 cells.
Sensitive neutralization assays
A sensitive neutralization assay was used to determine the neutralizing activity of hybridoma supernatants. Table 3 shows the percentage neutralization±SD of the different mAbs. The neutralizing activities of mAbs 13D12 and 1C11 were 7±19 and –1±14 %, respectively. As the mean neutralizing activity of mAb 13D12+SD was 7+19=26 %, a mAb was arbitrarily considered as neutralizing when its mean neutralizing activity was higher than 30 %. The 11 mAbs (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8) that reacted equally with all seven PCV-2 strains in the IPMA demonstrated neutralization of Stoon-1010 (up to 95 %), 48285 (up to 94 %), 1206 (up to 57 %) and 1103 (up to 61 %). The four mAbs (31D5, 48B5, 59C6 and 108E8) that had a higher affinity for genotype 2 strains than for genotype 1 strains in the IPMA demonstrated neutralization of the genotype 2 strains Stoon-1010 (up to 98 %) and 1103 (up to 67 %). For these four mAbs, neutralization of genotype 1 strains 48285 and 1206 was absent or very low (up to 35 %). mAb 13H4 did not neutralize any of the seven tested strains. Only one mAb (21C12) demonstrated some neutralization (32 %) of strain VC2002 and only two mAbs (9C3 and 38C1) demonstrated some neutralization (34 and 30 %, respectively) of strain 1147. None of the 16 mAbs neutralized strain 1121.
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) and a strain isolated from wild boars in Germany (accession no. AAU13781
[GenBank]
; Knell et al., 2005). Capsid protein similarity among the seven different strains used in this study was determined using pairwise alignments and CLUSTAL W (Fig. 2). The ORF2 amino acid identities of the strains used in this study is shown in Table 4. Fig. 3 shows a phylogenetic tree of the ORF2 protein based on the NJ method with the percentage confidence shown on each branch. This tree was constructed with ORF2 protein sequences from this study and sequences chosen from the different clusters from Olvera et al. (2007). The latter sequences are shown in Table 5. Genotype 1 strains 48285, 1206, VC2002-k39 and 1147 were assigned to cluster 1A/1B, VC2002-k2 to cluster 1C and genotype 2 strains Stoon-1010, 1121 and 1103 to cluster 2E. The same strain classification was obtained by using the ML method and with ORF2 DNA sequences.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Eleven mAbs (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8) reacted equally with the seven PCV-2 strains used in the IPMA. Four other mAbs (31D5, 48B5, 59C6 and 108E8) were able to differentiate the genotype 1 strains 48285, 1206, VC2002 and 1147 from the genotype 2 strains Stoon-1010, 1121 and 1103 by IPMA, as they did not react with genotype 1 strains or had a reduced affinity compared with genotype 2 strains. The IPMA results of the latter four mAbs were also reflected in the neutralization assays. Until now, mAbs have not allowed differentiation of PCV-2 strains (Allan et al., 1999; McNeilly et al., 2001). mAbs 31D5, 48B5, 59C6 and 108E8 also did not react with or had a reduced affinity for tissue sections originating from the Belgian PMWS-affected pig from which the VC2002 strain was isolated. This was demonstrated by immunofluorescence staining and suggests that the results obtained by IPMA for mAbs 31D5, 48B5, 59C6 and 108E8 were not a consequence of PCV-2 cell culture adaptation (data not shown).
Using the IPMA, mAbs 31D5, 48B5, 59C6 and 108E8 stained two different populations of infected cells in strain 1206. This suggests that strain 1206 consists of two viral subpopulations, where 99 % of the virus behaves as a genotype 1 strain and 1 % of the virus behaves as a genotype 2 strain. No signs of the existence of subpopulations were detected by sequencing of strain 1206. This may be explained by the fact that the putative genotype 2 subpopulation was present at a very low level (1 %). Sequencing of the VC2002 strain did reveal the existence of two PCV-2 subpopulations in the virus stock. After cloning, two distinct sequences were derived from strain VC2002. Phylogenetic analysis assigned clone VC2002-k39 to cluster 1A/1B and demonstrated clustering of clone VC2002-k2 with strains from China, The Netherlands (Grierson et al., 2004) and a strain isolated from German wild boars (Knell et al., 2005), which documents the putative epidemiological link between PCV-2 infections in domestic and wild pigs (Cságola et al., 2006). The identification of two different PCV-2 sequences in one animal has been reported previously (de Boisséson et al., 2004; Opriessnig et al., 2006; Cheung et al., 2007), but the role of multiple PCV-2 infections in the pathogenesis of PCV-2-associated diseases is not clear.
Using protein sequences (NJ and ML methods), we were not able to differentiate between clusters 1A and 1B, and not all sequences that were previously classified as 1C (Olvera et al., 2007) were found in the 1C cluster. Using the corresponding DNA sequences (NJ and ML methods), the same topology was obtained as by Olvera et al. (2007), with the only difference being that clusters 1A and 1B could not be differentiated in the present study (data not shown). We assume that these differences are a consequence of the reduced number of sequences used.
Putative amino acid substitutions that discriminate the genotype 1 strains 48285, 1206, VC2002-k39 and 1147 from the genotype 2 strains Stoon-1010, 1121 and 1103 are located at positions 63, 88, 89 and 206. At position 63, a threonine was substituted for a lysine or an arginine. At position 88, a lysine was replaced by a proline, and at position 89, an isoleucine was replaced by an arginine. These three substitutions all involve the basic amino acids lysine and arginine. Due to the differences in size, charge and hydrophobicity between lysine/arginine and threonine, proline and isoleucine, this may have major consequences on the secondary and tertiary structure of the PCV-2 capsid protein. The same applies to position 206, where a lysine was replaced by an isoleucine. Linear antigenic determinants of the PCV-2 ORF2 protein, as determined by PEPSCAN, are located at positions 65–87, 113–139, 169–183 and 193–207 (Mahé et al., 2000). Positions 63, 88 and 89, where non-conserved mutations were found in the present study, are located at the outer borders of linear epitope 65–87, whereas position 206, where another non-conserved mutation was found, is located at the inner border of linear epitope 193–207. Therefore, we speculate that the amino acid substitutions that involve basic amino acids at positions 63, 88, 89 and 206 might be responsible for the fact that mAbs 31D5, 48B5, 59C6 and 108E8 did not react with the genotype 1 strains or that they had a reduced affinity for these strains in the IPMA and neutralization assay.
This study also demonstrated that mAb 13H4 did not react specifically with the reproductive failure-associated strains 1121 and 1103 in the IPMA. Strains 1121 and 1103 have a proline at position 131 instead of a threonine (T131P) and an arginine instead of a glycine at position 191 (G191R). Proline is known to be a helix breaker and glycine has a great conformational flexibility. Apart from the changes in the primary structure of the protein, T131P and G191R may have important consequences on the secondary and tertiary structure of the protein. Position 131 is located within, and position 191 is located at the outer border of, an antigenic domain (Mahé et al., 2000). Therefore, the substitutions at positions 131 and 191 might be involved in the absence of reaction of mAb 13H4 with strains 1121 and 1103. Previously, it was demonstrated by Meerts et al. (2005a) that the production of infectious virus in PK-15 cells is more efficient for Stoon-1010 than for strain 1121. Fenaux et al. (2004) demonstrated that PCV-2 that was passaged 120 times in PK-15 cells (VP120) replicated more efficiently in PK-15 cells than wild-type virus that had been passaged only once (VP1). Differences between VP1 and VP120 were a mutation from proline to alanine at position 110 (P110A) and a mutation from arginine to serine at position 191 (R191S). This may suggest that basic amino acid residues at position 191 influence not only mAb reactivity, but also the production of infectious virus.
Recently, it was demonstrated that PMWS-affected animals are not able to produce neutralizing antibodies, whereas their ability to produce non-neutralizing antibodies remains unaffected (Meerts et al., 2005b, 2006; Fort et al., 2007). In these studies, it was suggested that PMWS-affected animals mount an immune response to non-neutralizing epitopes but not to neutralizing epitopes. In the present study, none of the tested mAbs was able to neutralize all seven PCV-2 strains, suggesting that a universal PCV-2 neutralizing epitope does not exist. Neutralization was observed for Stoon-1010, 48285, 1206 and 1103, but not VC2002, 1147 and 1121. No discriminative amino acid motifs that could explain these results were detected. The mAbs that neutralized Stoon-1010, 48285, 1206 and 1103 did not differentiate these strains from VC2002, 1147 and 1121 in the IPMA, indicating that these two different groups of PCV-2 strains have different neutralizing epitopes, and suggesting that these two different groups of PCV-2 strains use different entry pathways in PK-15 cells. Recently, the glycosaminoglycans heparan sulfate and chondroitin sulfate B have both been described as attachment receptors for PCV-2 (Misinzo et al., 2006). Protein binding to these two attachment receptors is restricted to the basic amino acids lysine and arginine (Esko, 1999), suggesting a crucial role of basic amino acid residues in the entry of PCV-2 into the host cell. The positive amino acid charges of lysine and arginine interact three-dimensionally with negatively charged glycosaminoglycan sulfates and carboxylates (Esko, 1999), which indicates that three-dimensional conformation plays a crucial role in interactions between the PCV-2 capsid protein and its receptors.
Until now, it was assumed that no distinct antigenic variation existed among PCV-2 isolates. In this study, we have clearly demonstrated the existence of major antigenic differences among the capsid proteins of PCV-2 strains with a different genotype and isolated from different clinical presentations.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 June 2007;
accepted 3 September 2007.
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