Agence Française de Sécurité Sanitaire des Aliments, Laboratoire de Biologie Moléculaire, BP 53, 22440 Ploufragan, France1
Author for correspondence: Dominique Mahé. Fax +33 2 96 01 62 83. e-mail d.mahe{at}ploufragan.afssa.fr
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Abstract |
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Introduction |
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Recently, a new situation has appeared with the emergence, in France and other European countries as well as in North America, of a new porcine disease known as post-weaning multisystemic wasting syndrome (PMWS). The disease is characterized clinically by fever and progressive weight loss, as well as respiratory and digestive disorders (Allan et al., 1998a , b
; Kennedy et al., 1998
; Kiupel et al., 1998
; Le Cann et al., 1997
; Morozov et al., 1998
; Nayar et al., 1997
; Segalés et al., 1997
). Morbidity rates ranging from 5 to 30% have been reported in affected batches of pigs (Madec et al., 2000
). Virus isolation from the tissues of pigs affected with PMWS led to the identification of PCV-like antigens and nucleic acids (Ellis et al., 1998
; Nayar et al., 1997
). These viruses were found to form a closely related group, exhibiting more than 96% sequence identity (Allan et al., 1998b
; Meehan et al., 1998
). However, they were significantly different from the PCV1 strain, thus representing a new type of porcine circovirus, which has been termed PCV2. Although they display similar genomic organization, their genomes share only about 70% sequence identity. Higher sequence conservation could be observed in the replicative ORF1-encoded protein (Rep protein).
PCV2s are believed to be of aetiological importance in PMWS (Allan et al., 1999a ; Balasch et al., 1999
; Ellis et al., 1999
) and the need for type-specific serological assays is therefore significant in examining the prevalence of PCV2 infection. The aim of the present study was to characterize the expression of the major viral proteins, to analyse their immunological properties and thereby to determine the extent of cross-reactivity between viral antigens from PCV types 1 and 2.
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Methods |
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PCR amplification and cloning of PCV genomes.
The PCV1 genome was cloned by utilizing an inverse PCR approach with forward (5' GGCGGCGCCATCTGTAACGGTTT 3') and reverse (5' GATGGCGCCGAAAGACGGGTATC 3') primers, each containing a NarI restriction endonuclease site (underlined). These primers were designed according to the PCV1 sequence presented in the accession number AF012107 (Mankertz et al., 2000 ) except that the forward primer was modified by two nucleotides in its 5' end without affecting the PCV1 sequence between the NarI sites. The PCV2 genome was cloned in a same inverse PCR approach by using forward (5' GTGGAGCTCCTAGATCTCAGGG 3') and reverse (5' TAGGAGCTCCACACTCCATCAG 3') primers deduced from the PCV2 sequence presented in the accession number AF201311 (Mankertz et al., 2000
). Each primer contains a SacI restriction endonuclease site (underlined).
Amplifications were performed with 0·4 µM of each primer, 0·1 mM of each dNTP and 2·5 U Taq DNA polymerase (Boehringer) with the following cycling parameters: denaturation at 94 °C, 5 min; 35 cycles (94 °C, 30 s; annealing at 60 °C, 30 s; 72 °C, 90 s); final elongation at 72 °C, 10 min. PCR products were then cloned into the pPCR-Script SK vector by using the pCR-Script Amp cloning kit (Stratagene). The clones were sequenced on both strands with an ABI 373 sequencer by using the M13 forward and reverse universal primers as well as circovirus genome-specific primers and the ABI Prism Dye terminator cycle sequencing kit (PE Biosystems).
Cloning of PCV genes.
The genes encoding ORF1 and ORF2 from both PCVs were amplified by PCR prior to cloning into the pcDNA3.1 mammalian expression vector (Invitrogen) and into either pVL1393 or pAcGHLT baculovirus transfer vectors (Pharmingen), the latter allowing the expression of glutathione S-transferase (GST)- and 6xHis-fused proteins. The following PCR primer pairs were used for PCV gene amplifications: for PCV1, ORF1 was amplified with ORF1.1 forward (5' CGGGATCCCAGTGAAAATGCCAAGCAAGAAAAG 3') and reverse (5' CGGGATCCCGATGTGATAACAAAAAAGACTCAG 3') primers, whereas ORF2 was amplified with ORF2.1 forward (5' CGGGATCCCTTTTTTGTTATCACATCGTAATGG 3') and reverse (5' CGGGATCCTCTTTCACTTTTATAGGATGACGTG 3') primers. For PCV2 gene amplification, ORF1.2 forward (5' ATGGATCCAGCAGACACATGCCCAGCAAG 3') and reverse (5' GGGGATCCGAAGTGATAAAAAAGACTCAG 3') primers were used to generate the ORF1 gene whereas ORF2 was amplified by using ORF2.2 forward (5' CCGGATCCTCCATTAAGGGTTAAGTGGG 3') and reverse (5' CCGGATCCTCAGATATGACGTATCCAAG 3') primers. All primers included a BamHI cloning site in their 5' end. Amplifications were performed as already mentioned except for the annealing temperature, which was 55 °C, and the elongation time, which was shortened to 45 s.
PCR products corresponding to the PCV2 genes were cloned into the pPCR-Script SK plasmid as indicated above. Recombinant clones were sequenced to ensure that the sequences did not contain mutations. The recombinant plasmids were then digested with BamHI. The purified fragment encoding the ORF1-PCV2 protein was subcloned into BamHI-cut pcDNA3.1 and pVL1393 plasmids, whereas the purified fragment encoding the ORF2-PCV2 protein was subcloned into BamHI-digested pcDNA3.1 and BglII-digested pAcGHLT.
For the PCV1 genes, PCR products were cloned directly into pcDNA3.1 vector after digestion with BamHI. Sequencing was performed for both constructs. The fragments encoding ORF1-PCV1 and ORF2-PCV1 were then purified and cloned into BamHI-digested pVL1393 and BglII-digested pAcGHLT, respectively.
Antisera
Anti-PCV2 antisera.
Lymph nodes obtained from PMWS-affected pigs and checked by PCR for the presence of PCV2 and the absence of PCV1 genomes were homogenized and ultrafiltered (0·45 µm) before intramuscular and intratracheal inoculations to either specific-pathogen-free (SPF) or conventional 6-week-old pigs. Sera were collected before virus inoculation and weekly for a maximum of 6 weeks post-infection (p.i.).
Hyperimmune sera.
Hyperimmune serum against ORF2-PCV2 was generated in SPF pigs that received two intramuscular injections of 200 µg of each pcDNA3.1 plasmid encoding ORF2-PCV2 and porcine granulocyte/macrophage colony-stimulating factor at 3 week intervals. Animals were then boosted 3 weeks later with 1 ml Sf9 cells (5x106) infected by a recombinant baculovirus encoding the ORF2-PCV2 protein and 1 ml Montanide adjuvant (Seppic). Blood was harvested 3 weeks after the last injection.
Polyclonal antibodies to ORF1-PCV1 were generated by immunizing mice (Agro-Bio). BALB/c mice received four injections of 50 µg of the corresponding recombinant pcDNA3.1 vector at 2 week intervals and two additional injections at 8 and 10 weeks.
Transfection/infection assays.
The permanent PK-15 cell line, which was free of PCV, was maintained in minimal essential medium (E-MEM, Biowhittaker) supplemented with 5% heat-inactivated foetal calf serum, penicillin and streptomycin. Cells were seeded in 24-well plates (3·5x104 cells per well) and grown to 6070% confluence. After one wash with OptiMEM medium (Gibco BRL), cells were transfected for 8 h with 0·1 µg DNA (either plasmids encoding viral genes or viral genomes) and 2·25 µg Transfectam reagent (Promega), according to the manufacturers protocol, except that OptiMEM was used for material dilution instead of serum-free E-MEM. For genome transfections, NarI-digested PCV1 and SacI-digested PCV2, obtained from the corresponding cloned genomes, were gel-purified and recircularized in the presence of T4 DNA ligase (Boehringer) overnight at room temperature before being transfected. Cells were then overlaid with complete medium and viral protein expression was analysed by an immunoperoxidase monolayer assay (IPMA) as described below, 48 h after gene transfections or 72 h after genome transfections. In the case of genome transfections, cells were additionally treated 24 h after transfection with 300 mM D-glucosamine as described previously (Tischer et al., 1987 ).
For the infection test, genome-transfected cells were subjected to three successive freezethaw cycles. Following a centrifugation step at 2000 g for 5 min, lysate supernatants were collected and used to infect PCV-free PK-15 cells. Freshly trypsinized cells were seeded into 96-well plates and, once semi-confluent, they were inoculated for 1 h at 37 °C with lysate supernatant. They were then subjected to glucosamine treatment as described above and analysed by IPMA 72 h after infection.
For IPMA analysis, cells were fixed and permeabilized at -20 °C with 80% cold acetone and washed with PBS. The cells were then incubated for 1 h at 37 °C with 4% dry milk (Bio-Rad) in PBS containing 0·05% Tween 20 (PBS/Tw) and 0·1% Triton X-100 and incubated for a further 90 min at 37 °C with antisera diluted 100 times in PBS/Tw containing 2% dry milk. After three washes with PBS containing 0·25% Tween 20, cells were incubated for 90 min at 37 °C with HRP-conjugated rabbit anti-mouse or anti-swine IgG (Dako) in PBS/Tw containing 2% dry milk and washed again. Staining was developed in the presence of 9-aminoethyl carbazole (Sigma) in dimethylformamide as well as hydrogen peroxide and was stopped by substrate removal.
Generation and production of recombinant baculoviruses.
Sf9 cells, maintained in TC100 medium (Gibco BRL) supplemented with 5% foetal calf serum, were co-transfected with 500 ng linearized Baculogold baculovirus DNA (Pharmingen) and 5 µg recombinant baculovirus transfer vector by using the DOTAP transfection reagent (Boehringer) according to the suppliers instructions. Recombinant baculoviruses were plaque-purified and amplified.
Expression and identification of recombinant proteins.
Sf9 cells were infected with wild-type Autographa californica multiple nuclear polyhedrosis virus or recombinant baculoviruses at an m.o.i. of 10 p.f.u. per cell. The cells were harvested 72 h p.i. and protein expression was analysed by immunoblot. Total proteins from 105 cells were then separated on a 10% SDSpolyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad). The proteins were identified by using either an anti-GST polyclonal antibody (Pharmingen) or an anti-PCV antiserum and a peroxidase-labelled rabbit anti-mouse or anti-swine IgG conjugate (Dako).
PEPSCAN analysis.
The simultaneous synthesis of peptide sequences was carried out on a cellulose membrane by Fmoc amino acid chemistry (SYNT:EM). Overlapping peptides covering the last 214 and 213 amino acids, respectively, of ORF1 from PCV1 and PCV2 (102 peptides) as well as the complete sequence of ORF2 (112 peptides) and ORF3 (73 peptides) from both PCVs were synthesized on the membrane. The peptides, which were 15 amino acids in length, overlapped by 11 amino acids and differed by a four amino acid shift. Antibody reactivity to the membrane-bound peptides was analysed by an indirect colorimetric immunoassay according to the manufacturers recommendations (SYNT:EM).
Briefly, the membranes were incubated overnight in blocking buffer at 4 °C. After washing once in 50 mM TrisHCl, pH 7, containing 150 mM NaCl, 30 mM KCl and 0·05% Tween 20, the membranes were incubated with serum diluted 100 times in blocking buffer. Development was performed in the presence of BCIP, MTT and MgCl2. Spots corresponding to peptides with antibody reactivity produced a positive blue signal, which was quantified by using the NIH 3.1 Image software.
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Results |
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Discussion |
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By using ORF1-PCV1 hyperimmune serum, we were able to demonstrate that the ORF1 proteins were antigenically related in the two PCV types. This was also confirmed by using recombinant ORF1 proteins from PCV1 and PCV2, which were both recognized by anti-PCV2 antisera. The presence of anti-ORF1 antibodies after PCV2 infection was also shown in this way. In contrast, the absence of cross-reaction that was reported by Balasch et al. (1999) with experimental anti-PCV2 sera indicated the absence of anti-ORF1 antibodies. This could be explained by the time after experimental infection that the sera were collected. Indeed, ORF1 antibodies, which would be absent or present at a very low titre at least up to 2 weeks p.i. (Balasch et al., 1999
), might appear later after infection (this study).
Further analysis of the antigenic regions of ORF1-encoded proteins with synthetic peptides showed that, in the last 200 amino acids, a common antigenic determinant could be detected between residues 185 and 211 of the proteins. Furthermore, since the first 100 amino acids, which display strong identity between the two proteins, were excluded from this study, we cannot exclude the presence of common antigenic domains in this area. In contrast, no discriminating epitope could be identified. The demonstration of serological cross-reactivity between the ORF1 proteins correlates well with the high degree of sequence identity observed between the two types of PCV and is fully consistent with its essential conserved function for virus replication (Mankertz et al., 1998a ).
Immunoreactivity against the putative capsid protein encoded by the major ORF2 was also investigated. In contrast to ORF1, a hyperimmune serum generated against ORF2 from PCV2 did not allow the detection of viral protein in PCV1-transfected cells. Hence, the question was whether this absence of signal resulted from the lack of serological reaction between ORF2-PCV2 antiserum and ORF2-PCV1 protein, since it could also be explained by the absence of ORF2 expression by PCV1. The identification of ORF2 transcript expression in PCV1-infected cells (Mankertz et al., 1998b ) does not support this latter hypothesis. Moreover, ORF2 proteins fused to GST were expressed and used to demonstrate here that, in spite of their 56% sequence identity, no detectable cross-reactivity could be shown between ORF2 proteins. Antigenic differences have already been reported between PCV1 and PCV2 isolates, as demonstrated by the differences in anti-PCV antibody titres on infected cells. These differences might thus be explained by the differential antigenicity of ORF2-encoded structural proteins (Allan et al., 1999b
; Ellis et al., 1998
). Further analysis of immunodominant regions determined by using ORF2 synthetic peptides led, as was expected, to the identification of antigenic domains in only the ORF2-PCV2 protein (residues 6587, 113139 and 193207). However, a few reactive peptides common to ORF2 from PCV1 and PCV2 could also be identified in this way, although variations could be observed depending on the serum used. The absence of cross-reactivity observed in transfected cells or after immunoblot assays might then be explained by the lack of accessibility of this peptide sequence in the whole protein.
The differential immunoreactivity of ORF2-PCV2 peptides or proteins with anti-PCV2 antibodies is of particular interest, since such reagents could be used to develop an immunoassay such as ELISA for the detection of PCV2 infection in herds. Of the reactive peptides chosen to represent the antigenic domains of ORF2 protein, one, B-133, could be selected for such ELISA development, since it allowed anti-PCV2 antibody detection and discrimination in both SPF and farm antisera (unpublished results). This peptide shows 25% amino acid sequence divergence from its counterpart, the A-189 peptide.
As demonstrated by PEPSCAN analysis, greater immunoreactivity was observed for the ORF2 protein, which encodes the putative capsid protein. A sequence comparison of this protein from the different strains of PCV2 revealed some variations in the selected peptides. Among them, the B-133 peptide sequence differs by one amino acid between European and North American strains. Whether the anti-PCV2 antiserum generated from a French PCV2 strain could recognize the same epitopes in strains from other countries is not yet known and would provide information about epitope variability among PCV2 strains.
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Acknowledgments |
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References |
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Received 25 January 2000;
accepted 22 March 2000.