Department of Veterinary Microbiology and Preventive Medicine1, Department of Veterinary Diagnostic and Production Animal Medicine2, and Veterinary Medical Research Institute (VMRI)3, Iowa State University, 1802 Elwood Drive, Bldg 6, Ames, IA 50011-1240, USA
Viral and Prion Diseases of Livestock Research Unit, National Animal Disease Center, ARS, USDA, Ames, Iowa, USA4
Author for correspondence: Prem Paul. Fax +1 515 294 1401. e-mail pspaul{at}iastate.edu
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Abstract |
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Introduction |
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Recently, PCV was found associated with the newly emerged post-weaning multisystemic wasting syndrome (PMWS) of swine (Allan et al., 1998 ; Ellis et al., 1998
; Meehan et al., 1998
; Morozov et al., 1998
). Manifestations of PMWS include unthriftiness, respiratory distress and jaundice (Harding, 1997
). Frequently encountered microscopic lesions are interstitial pneumonia, lymphoid depletion and hepatitis (Clark et al., 1997
). Monoclonal antibodies raised against PCV in the PK-15 cell line have been used to differentiate that virus from PCV associated with PMWS (Allan et al., 1998
). Genetic analysis clearly indicates that two genotypes of PCV exist (Hamel et al., 1998
; Meehan et al., 1998
; Morozov et al., 1998
). PCV in the PK-15 cells represents type 1 (PCV1), and PCV associated with PMWS segregates into a second virus genotype, PCV2 (Meehan et al., 1998
).
To date, structural protein(s) of PCV2 and the open reading frame(s) (ORF) that code for viral structural protein(s) have not been identified. In this study, we identified a protein of 30 kDa as a viral structural protein which is encoded by ORF2 of PCV2. This protein independently forms viral capsid-like structures when expressed in insect cells from recombinant baculovirus.
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Methods |
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Viruses.
To produce purified virus, PCV2 strain ISU31 (Morozov et al., 1998 ) was propagated in the PK-15 cell line as described by Tischer et al. (1986)
. The infected cells were frozen and thawed three times, clarified at 27000 g for 30 min and pelleted at 270000 g for 6 h. The pellet was resuspended in NET buffer (10 mM Tris pH 7·4, 100 mM NaCl, 1 mM EDTA and 10 mM MgSO4) and centrifuged in a solution of 37% CsCl (w/v) in water to equilibrium for 48 h at 270000 g. A readily visible opaque band was collected by aspiration, washed and dissolved in NET buffer.
To make PCV2 stock, purified PCV2 strain ISU31 was inoculated onto a semi-confluent PCV-free PK-15 cell line according to the method of Tischer et al. (1986) . Briefly, the infected cells were treated with 300 mM D-glucosamine (Sigma) for 30 min at 37 °C at 4 h post-inoculation (p.i.). When the cells were confluent, they were trypsinized and passaged. The cells were treated with D-glucosamine (300 mM) 1 day after subculture. When 80% of cells were showing cytopathic effect, characterized by rounding and fragmentation of cells, the infected cells were frozen and thawed three times and the resulting suspension of cell debris and membrane was clarified by centrifugation. The viruses in the supernatant were pelleted at 270000 g for 6 h. The virus pellet was treated with an equal volume of Freon (Fisher Scientific) and filtered through a 0·2 µM filter.
Wild-type (wt) Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant AcMNPV expressing ORF2 (AcMNPV.ORF2) stocks were prepared as previously described (OReilly et al., 1992 ). To prepare working stock of wild-type and recombinant baculoviruses, wt AcMNPV and AcMNPV.ORF2 were propagated in Sf9 monolayer cultures for 5 days. The infected cells and supernatant were frozen and thawed three times and clarified by centrifugation at 1100 g for 10 min.
Molecular cloning and expression of viral genes.
A Bac-to-Bac baculovirus expression system (Gibco BRL) was used for expression of the ORF2 gene. Viral DNA was isolated from PK-15 cells infected with the ISU31 strain of PCV2. ORF2 of PCV2 strain ISU31 was amplified by PCR using primers 5' AGT GCT CGA GGG ATC CAT GAC GTA TCC AGG GAG GCG 3' and 5' GAG CAG ATC TTT AGG GTT TAA GTG GGG GGT CTT TAA G 3' (sequence specific for PCV2 ORF2 is in bold type; sequence engineered to create cloning sites is in roman type). The ORF2 fragment was cloned into XhoI and BglII sites of plasmid pKSII+ (Stratagene). The integrity of the base sequence of ORF2 in pKSII+ was verified by sequence analysis. To produce the donor recombinant plasmid (pPSP.PCV.ORF2) for derivation of recombinant baculovirus, the ORF2 gene was subcloned into pFastbac (Gibco BRL) at BamHI and SpeI restriction sites. Recombinant baculovirus carrying the ORF2 gene was constructed according to the manufacturers instructions (Bac-to-Bac baculovirus expression system, Gibco BRL). Briefly, E. coli DH10Bac (Gibco BRL) containing baculovirus shuttle vector (bacmid) and helper vector was transformed with recombinant plasmid pPSP.PCV.ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid were collected by blue/white selection. The recombinant bacmid DNA was isolated, purified and transfected into Sf9 cells to yield AcMNPV carrying the PCV2 ORF2 gene, referred to as AcMNPV.ORF2, under the control of the polyhedrin promoter. Primary stock of AcMNPV.ORF2 was harvested at 72 h post-transfection. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay using hyperimmune serum raised against PCV2 in rabbits (Sorden et al., 1999 ).
Purification of recombinant ORF2 expression product.
Purified ORF2 expression protein was obtained from lysates of ORF2 gene product. Sf9 cells infected with AcMNPV.ORF2 were lysed at 72 h p.i. according to the method of Wong et al. (1994) and purified by CsCl gradient centrifugation as described for PCV2. Briefly, AcMNPV.ORF2-infected Sf9 cells were lysed in buffer containing 50 mM sodium borate, 150 mM NaCl, 1% Nonidet P-40, 0·5% sodium deoxycholate and 5%
-mercaptoethanol. The lysates were diluted in PBS, laid on top of 40% sucrose in PBS and centrifuged at 270000 g for 6 h. The pellet was resuspended in NET buffer and centrifuged to equilibrium in 37% CsCl in water at 270000 g for 48 h. The opaque band in the middle of the tube was collected and dialysed in Tris buffer (50 mM Tris and 150 mM NaCl, pH 7·2) for 24 h with three changes of the buffer. Sf9 cells and wt AcMNPV-infected Sf9 cells were lysed and purified by a method similar to that used for AcMNPV.ORF2-infected Sf9 cells.
Cell lysates.
The PCV2 purified by CsCl gradient centrifugation was lysed with an equal volume of Laemmli sample buffer (Bio-Rad) and boiled for 5 min. To prepare cell lysates from PCV2-infected cells, 25 cm2 flasks of semi-confluent PCV-free PK-15 cells were inoculated with PCV2 stock at 1 m.o.i. Control lysates were prepared from mock-infected PK-15 cells. At 60 h p.i., the medium was removed and replaced with 1 ml Laemmli sample buffer (Bio-Rad). The cells were incubated with the sample buffer for 2 min on ice before they were scraped, transferred to a microfuge tube and boiled for 5 min.
To prepare lysates from baculovirus-infected cells, wt AcMNPV and AcMNPV.ORF2 were inoculated onto monolayers of Sf9 cells at an m.o.i. of 5. Mock-infected Sf9 cells were used to prepare control cell lysates. The cells were lysed at 72 h p.i. as described previously. Recombinant ORF2 expression protein lysate was prepared from the purified ORF2 protein, containing virus-like particles, which was dissolved in an equal volume of Laemmli sample buffer (Bio-Rad), boiled for 5 min and used for electrophoresis.
Preparation of anti-PCV sera.
Rabbit anti-PCV2 hyperimmune serum was prepared as described elsewhere (Sorden et al., 1999 ). The gradient-purified PCV2 was diluted in 0·85 % NaCl to a concentration of 1 mg/ml and mixed with adjuvant MLP+TPM+CWS (Sigma). The rabbits were inoculated with PCV2adjuvant emulsion. Immunizations were repeated a total of three times at 3-week intervals. The serum was collected 2 weeks after the final immunization and tested for the presence of antibodies to PCV2 by indirect immmunofluorescent assay. The serum was absorbed with PK-15 cells or Sf9 cells as described by Harlow & Lane (1988)
.
Samples of swine serum were obtained from a pig experimentally inoculated with strain 35358 of PCV2 at 0, 7, 14, 21, 28, 36, 42 and 49 days p.i.
Western blot.
SDSPAGE was done according to the method of Laemmli (1970) using a 1 mm thick 15% slab gel. After electrophoresis, proteins were transferred onto nitrocellulose membranes (Bio-Rad) using a mini Trans-Blot transfer cell (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine, 20%, v/v, methanol) at 100 V for 90 min. Immunoblots were performed as described by Harlow & Lane (1988)
and Zhang et al. (1998)
with slight modification. The nitrocellulose membrane was soaked in 0·5% blocking solution (Roche) before incubation with rabbit anti-PCV2 serum (1:3000) or swine anti-PCV2 serum (1:100) overnight at 4 °C. The blots were reacted with peroxidase-labelled anti-rabbit or anti-swine IgG (KPL) for 1 h at room temperature. The membranes were washed five times with Tris buffer saline (50 mM Tris pH 7·5, 200 mM NaCl) containing 0·1% Tween 20 between each incubation. After equilibration in 0·15 M Tris pH 9·5, bound antibodies were detected with 3,3',5,5'-tetramethylbenzidine (TMB) substrate peroxidase solution (KPL).
Negative staining electron microscopy and immunoelectron microscopy.
Purified PCV2 and purified ORF2 expression product from the CsCl gradient centrifugation were allowed to absorb onto carbon-coated copper grids for 7 min. Then, the grids were dried using filter paper and negatively stained with 3% phosphotungstic acid (PTA) for 20 s for purified PCV2 particles and for 5 min for the expression product from ORF2. The samples were examined at a magnification of 35000x using a Hitachi H500 transmission electron microscope.
For immunoelectron microscopy, the CsCl gradient-purified recombinant ORF2 protein was incubated with either 1:500 dilution of rabbit anti-PCV2 hyperimmune serum or 1:500 dilution of preimmune rabbit serum for 45 min at 37 °C. The proteinserum mixtures were allowed to adsorb onto carbon-coated copper grids by agar diffusion for 30 min. The grids were stained with 3% PTA for 2 min and viewed using the transmission electron microscope as previously described.
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Results |
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Detection of antibodies against ORF2 recombinant protein in swine serum
To determine the antigenic authenticity of the expression product from ORF2, samples of serum obtained from swine at periodic intervals after experimental infection with PCV2 were used for immunoblotting. Blots were prepared from lysates of Sf9 cells infected with recombinant AcMNPV.ORF2 and incubated with swine sera obtained at 0, 7, 14, 21, 28, 35, 42 and 49 days p.i. The results showed that antibodies against the ORF2 expression product were detected as early as 21 days after infection (Fig. 5). The intensity of the signal on the Western blot indicated that the antibody titre increased in serum over time after challenge exposure of pig with virus.
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Discussion |
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Both ORF1 and ORF2 have a theoretical coding capacity of proteins of 28 kDa or larger (Hamel et al., 1998 ; Meehan et al., 1998
; Morozov et al., 1998
). The predicted amino acid sequence of ORF1 from either PCV1 or PCV2 revealed at least three amino acid motifs found in the Rep protein which are associated with rolling circle replication (Mankertz et al., 1997
; Meehan et al., 1997
, 1998
; Morozov et al., 1998
). Additionally, a plasmid containing ORF1 of PCV1 enhanced replication of the PCV1 origin of replication, confirming that ORF1 of PCV1 encodes the Rep protein (Mankertz et al., 1998a
). On the other hand, the predicted amino acid sequence of ORF2 from either PCV1 or PCV2 contained a conserved region of basic amino acids at the N-termini, similar to that observed for the major structural protein of chicken anaemia virus (Meehan et al., 1998
). For PCV1, transcription analysis indicated that the ORF2 transcript was the most abundant (Mankertz et al., 1998b
), corresponding to our finding of a high level expression of the 30 kDa protein in PCV2-infected PK-15 cells. The predicted molecular mass of the translated product of ORF2 is 28 kDa (Hamel et al., 1998
; Meehan et al., 1998
; Morozov et al., 1998
), which is close to the estimated 30 kDa expression product of ORF2 reported here.
Our studies show that ORF2 encodes a 30 kDa protein that is involved in viral capsid formation. Virus-like particles formed by the expression product of ORF2 were similar to intact PCV particles but some of the self-assembled virus-like particles appeared empty. These findings are consistent with those reported for several non-enveloped viruses, such as parvovirus (Brown et al., 1991 ; Christensen et al., 1993
), polyomavirus (An et al., 1999
), calicivirus (Geissler et al., 1999
; Prasad et al., 1999
) and hepatitis E virus (Xing et al., 1999
). Expression of the major structural protein of each of the aforementioned viruses gave rise to the formation of empty capsids (Brown et al., 1991
; An et al., 1999
). Some of the ORF2 self-assembled particles observed in our study were less ordered than those in the purified PCV2 preparation. A similar finding was reported for the mild truncated minor capsid protein of parvovirus B19 (VP1) expressed in baculovirus, and probably resulted from improper assembly (Wong et al., 1994
). The major capsid protein (VP2) of parvovirus B19 is the truncated version at the N terminus of its minor structural protein counterpart (VP1) and both are transcribed from the same gene (Shade et al., 1986
; Ozawa & Young, 1987
). In the case of PCV2, whether this morphology results from the lack of minor structural protein(s) or a requirement of DNA for perfect assembly needs to be further investigated.
Antibodies raised against the ORF2 gene product could be clearly detected as early as 21 days after infection. These antibodies persisted for 49 days (the duration of experiment). These studies suggest a potential use of recombinant 30 kDa protein for the detection of PCV infection in pigs and as a vaccine.
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Acknowledgments |
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References |
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Received 13 March 2000;
accepted 7 June 2000.