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Journal of Clinical Microbiology, September 1998, p. 2535-2541, Vol. 36, No. 9
Veterinary Medical Research
Institute,1
Department of Veterinary
Pathology,2
Department of Veterinary
Diagnostic and Production Animal Medicine,3 and
Department of Microbiology, Immunology and Preventive
Medicine,4 College of Veterinary Medicine,
Iowa State University, Ames, Iowa 50011
Received 12 January 1998/Returned for modification 23 April
1998/Accepted 4 June 1998
Swine infectious agents, especially viruses, are potential public
health risks associated with the use of pig organs for
xenotransplantation in humans. Therefore, there is a need for better
characterization of swine viruses and for the development of
diagnostic tests for their detection. We report here isolation of a
novel strain of porcine circovirus (PCV) from pigs with postweaning
multisystemic wasting syndrome (PMWS). Affected pigs exhibited severe
interstitial pneumonia and lymphoid depletion. The complete nucleotide
sequence (1,768 nucleotides) of the genome of the PCV isolate was
determined and compared with the sequence of the PCV strain isolated
from PK-15 cells. Sequence comparison revealed significant differences between the two PCV strains, with an overall DNA homology of 76%. Two
major open reading frames (ORFs) were identified. ORF1 was more
conserved between the two strains, with 83% nucleotide homology and
86% amino acid homology. ORF2 was more variable, with nucleotide homology of 67% and amino acid homology of 65%. PCR and in situ hybridization demonstrated abundant viral DNA in various organs of pigs
with PMWS. In situ hybridization demonstrated that this strain of PCV
targets multiple organs and infects macrophages, lymphocytes,
endothelial cells, and epithelial cells.
The infectious agents of swine are
receiving increased attention due to the potential use of pig organs
for xenotransplantation in humans. Some of these agents are poorly
studied, and/or their significance to swine health is unknown. Porcine
circovirus (PCV) is one such agent and is considered to be widespread
in swine (7, 33, 35). PCV is a member of the family
Circoviridae, which consists of DNA viruses with a circular,
single-stranded genome. Other members include chicken anemia virus and
psittacine beak and feather disease virus (28, 38) in
animals and several plant viruses, including subterranean clover stunt
virus, coconut foliar decay virus, and banana bunchy top virus (4,
15, 29). PCV was first isolated in 1974 as a persistent
contaminant of the continuous porcine kidney cell line PK-15 (ATCC
CCL31) (34, 37), and the PCV strain isolated from PK-15
cells (PCV PK-15) has been well characterized (3, 20, 23).
No common antigenic determinants or DNA sequence homologies among
animal circoviruses have been detected (39).
Serological surveys using indirect immunofluorescence or
immunoperoxidase assays indicate that antibodies to PCV PK-15 are very common in North American and European swine herds. Antibodies to
PCV were found in 85% of the sera of slaughter pigs in Germany (35), in 53% of the samples in a survey of swine in the
United States (18), in 86% of randomly collected serum
samples in Britain (8), and in 92% of randomly collected
serum samples in Ireland (3). A survey conducted with
an enzyme-linked immunosorbent assay in Germany showed that in
almost all swine herds tested (with one exception), PCV infection was
common (33). Antibodies reacting with PCV PK-15 have
also been detected in humans, mice, and cattle (32).
Despite the common occurrence of PCV infection, the clinical
significance of PCV in swine and other species is not known. Limited
experimental transmission studies suggest that the PCV PK-15 strain may
not cause any overt disease in swine (2). However, several
reports suggesting that PCV is associated with postweaning
multisystemic wasting syndrome (PMWS) in growing pigs (6, 13, 14,
27), and possibly with congenital tremors in newborn pigs
(17), have been published recently. PMWS is a newly emerged
porcine disease syndrome, characterized clinically by progressive
dyspnea, emaciation, and lymph node enlargement and pathologically by a
wide range of inflammatory lesions that most often include
lymphohistiocytic to granulomatous lymphadenitis, interstitial
pneumonia, hepatitis, interstitial nephritis, and pancreatitis (6,
13, 14).
The purpose of this study was to determine whether PCV isolated from
pigs with PMWS is related to PCV PK-15, as well as to develop PCR and
in situ hybridization assays for the detection of PCV in clinical
samples. Such methods should be useful in elucidating the role of PCV
in swine disease syndromes and for providing PCV-free xenografts. This
report describes the isolation and genetic characterization of a new
PCV strain (PCV ISU-31) from tissues of pigs with PMWS which is
significantly different from the PCV strain previously isolated from
the PK-15 cell line.
Clinical history and tissue samples.
A group of 1,200 pigs
weighing 75 to 85 lb in an eastern Iowa grower-finisher herd had a 3 to
4% incidence of dyspnea, depression, and ill thrift. These pigs had
been farrowed in Canada and weaned at 14 to 21 days, transported to a
nursery unit in northern Iowa, and moved to the grower-finisher herd
when they weighed 50 to 60 lb. Affected pigs were minimally responsive
to treatment with various antibiotics. Microscopic examination of
tissues from one dead pig submitted to the Iowa State University
Veterinary Diagnostic Laboratory revealed severe lymphohistiocytic
interstitial pneumonia and severe depletion of tonsilar and splenic
lymphoid tissue. Moderate numbers of macrophages in the tonsil
contained intracytoplasmic clusters of globular amphophilic inclusion
bodies. Immunohistochemistry revealed porcine reproductive and
respiratory syndrome virus (PRRSV) antigen in the lung, and PRRSV was
isolated from pooled lung, tonsil, and lymph node. PRRSV
immunohistochemistry and virus isolation were performed as described
previously (10, 12). Findings for this pig were consistent
with both PMWS and porcine reproductive and respiratory syndrome
(PRRS), and additional clinically affected pigs (five live and one
dead) were obtained from the same herd for further studies. Five of the
six pigs had microscopic lesions consistent with PMWS.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of a Novel Strain of Porcine Circovirus
in Pigs with Postweaning Multisystemic Wasting Syndrome
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
70°C. Total
DNA was isolated from 25 mg of each tissue sample and used for PCR
experiments. For histopathologic studies, samples were fixed in 10%
buffered formalin, routinely processed, and embedded in paraffin.
Five-micrometer-thick sections for histologic examination and in situ
hybridization were cut from the same blocks. Samples of lung, liver,
and lymph node stored at 70°C were used for virus isolation.
DNA isolation, PCR amplification, cloning, and sequencing. DNA was isolated from lung, liver, spleen, intestine, lymph node, and tonsil samples of animals with PMWS. The DNA was isolated from 25 mg of each tissue sample with a QIAamp Tissue Kit (Qiagen, Santa Clarita, Calif.). PCR amplification was performed with 100 ng of DNA in a reaction mixture with five different sets of primers designed on the basis of the sequence of the PCV PK-15 strain. As a negative control for PCR, DNA isolated from PK-15 cells was used. PCR was performed in 30 cycles with the following parameters: denaturation at 94°C for 20 s, annealing at 61°C for 20 s, and elongation at 72°C for 45 s. PCR products were analyzed by agarose gel electrophoresis. Only in PCR with primers PCVF (5'-CAGCGGCAGCACCTCGGCAGCGTCAGT-3') and PCV507 (5'-TCCAATCACGCTGCTGCATCTTCCCGC-3') was a specific 530-bp PCR product amplified from tissue DNA of all samples tested. The PCR product amplified from the lymph node of one animal was isolated, sequenced, and cloned into a pGEM-T vector (Promega, Madison, Wis.). The resulting plasmid, pPSP.PCV1, was used for in situ hybridization. Two specific primers, PCV75.1 (5'-CGGAAGGATTATTCAGCGTGAACACCC-3') and PCV389 (5'-GCTGTGAGTACCTTGTTGGAGAGCGGG-3'), were synthesized and used to amplify the rest of the genome of this PCV isolate, referred to as PCV ISU-31, from the same DNA sample. The 1.5-kb DNA fragment was amplified and sequenced. Sequencing data were analyzed with the GeneWorks (IntelliGenetics, Inc., Mountain View, Calif.) and MacVector (International Biotechnologies, Inc., New Haven, Conn.) DNA analysis programs. During virus isolation, total DNA was isolated from the monolayer of experimentally infected and noninfected PK-15 cells with the QIAamp Tissue Kit (Qiagen) and used in PCR with the primers described above.
In situ hybridization. Lymph node, spleen, tonsil, liver, lung, heart, kidney, pancreas, nasal turbinate, and large and small intestine samples were fixed in 10% buffered formalin and processed for in situ hybridization as described previously for coronaviruses (31), with slight modifications. The plasmid pPSP.PCV1, linearized with restriction enzyme NcoI (Stratagene, La Jolla, Calif.), was used as a template to make an antisense fluorescein-labeled RNA probe. In order to make a sense RNA probe, plasmid pPSP.PCV1 was linearized with restriction enzyme PstI (Stratagene), and RNA was synthesized with T7 RNA polymerase (Stratagene). In situ hybridization was performed as described previously (31) with slight modification. Sections of 5-µm thickness were deparaffinized, rehydrated, and treated with proteinase K (50 µg/ml). Before hybridization, tissues were incubated with 200 µl of hybridization buffer by using a CoverWell chamber (Grace Bio-Labs, Bend, Oreg.) at 95°C for 10 min. Sections were hybridized, washed, and treated with RNase A. Then, sections were incubated with anti-fluorescein alkaline phosphatase, washed, incubated with 4-nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (Boehringer Mannheim, Indianapolis, Ind.), and counterstained with nuclear fast red. Tissues from two negative control pigs, two pigs experimentally infected with PRRSV, and nondenatured RNase-treated PMWS tissues hybridized with the sense RNA probe served as negative controls.
Virus isolation. Tissues from lung, liver, and lymph node of the pig with PMWS from which PCV ISU-31 was PCR amplified were pooled and homogenized in minimal essential medium containing penicillin (100 U/µl) and streptomycin (100 µg/ml) to make an approximately 10% suspension. The sample was clarified, filtered through a 0.22-µm-pore-size filter, and used as an inoculum for further studies. Virus isolation was carried out in PK-15 cell culture shown by PCR to be free of PCV PK-15 contamination. Two 75-cm2 flasks of semiconfluent PK-15 cells were inoculated with 3 ml of inocula and incubated at 37°C in CO2 atmosphere for virus adsorption. Then, 15 ml of minimal essential medium supplemented with 10% fetal bovine serum and antibiotics was added, and flasks were incubated for 4 h more. Following incubation, cells were treated with 300 mM D-glucosamine (36) for 30 min and then incubated for a further 48 h. One flask was used for total DNA isolation, and another flask was passaged into fresh 75-cm2 tissue culture flasks for further propagation. Cells were passaged every 2 days and treated with D-glucosamine 24 h after each passage. After passages 1 to 4, total DNA was isolated from infected and noninfected cells and tested by PCR for the presence of PCV DNA, as described above. Infected cells were also tested by indirect immunofluorescence assay (IIFA) with polyclonal serum collected from pigs with clinical signs of PMWS. For IIFA, infected cells were distributed into 12-well cell culture plates and fixed with cold methanol 24 h after glucosamine treatment. After fixation, cells were washed twice with phosphate-buffered saline (PBS) and incubated with primary polyclonal serum (1:10 dilution in PBS) at 37°C for 1 h. The cell cultures were washed twice with PBS and immunostained with a 1/50 dilution of fluorescein isothiocyanate-conjugated goat anti-swine immunoglobulin G antibodies (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) in PBS at 37°C for 30 min. After a wash with PBS, cells were examined with a fluorescence microscope for the presence of specific fluorescent staining.
Nucleotide sequence accession number. The EMBL nucleotide sequence database accession number of the sequence reported in this paper is AJ223185.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Gross and histopathologic findings. Six of the seven pigs examined in this study had lesions consistent with PMWS. Lungs were noncollapsed, rubbery, and mottled red to pale tan. Inguinal, tracheobronchial, and mesenteric lymph nodes were moderately to markedly enlarged and pale. Microscopic examination revealed severe lymphohistiocytic interstitial pneumonia with mild to marked type 2 pneumocyte hyperplasia. Alveoli contained neutrophils, eosinophils, macrophages, and fibrinonecrotic debris. In some areas there was marked multifocal fibrosis of airway laminae propriae and bronchiolitis obliterans. Peyer's patches, tonsils, and spleens exhibited marked depletion of lymphocytes and variable infiltrates of macrophages and fewer multinucleate giant cells. Predominantly in the Peyer's patches, occasional macrophages within lymphocyte-depleted foci contained multiple globular basophilic to amphophilic intracytoplasmic inclusion bodies. Infiltrates of lymphocytes and macrophages were inconsistently present within liver, pancreas, kidney, and gastric tunica muscularis samples. Immunohistochemistry revealed PRRSV antigen in the lungs of two of the pigs. Typical histological lesions observed in lymphoid tissues of pigs with PMWS are shown in Fig. 1.
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PCR amplification and genetic characterization of a new PCV isolate. Because the intracytoplasmic clusters of basophilic inclusions were consistent with PCV infection, DNA was isolated from lung, liver, spleen, intestine, lymph node, and tonsil samples of animals with lesions of PMWS. Circovirus DNA was successfully amplified from all tested samples. From one sample, the entire genome of PCV was amplified in two overlapping fragments, cloned, and sequenced.
Analysis of sequencing data showed that PCV ISU-31 has a 1,768-bp DNA genome. The genome of PCV ISU-31 was 9 nucleotides longer than the genome of PCV PK-15. Significant differences were found between these two strains, with an overall DNA homology of 76%. Two major open reading frames (ORFs), ORF1 and ORF2, located in opposite orientations, were detected in the genome of PCV ISU-31. Two noncoding regions, 83 and 44 bp long, were located between ORF1 and ORF2. ORF1 in the PCV ISU-31 genome is 942 bp long and is predicted to encode a protein of 314 amino acids. In comparison, ORF1 of PCV PK-15 is 936 bp long, with a predicted protein of 312 amino acids. The homology in ORF1 between these two strains was 83% at the nucleotide level and 86% at the amino acid level. Figure 2A shows the alignment of the predicted ORF1 proteins of PCV ISU-31 and PCV PK-15, with three variable regions. Variable region 1 is at the 5' end of the protein, where there is a 3-amino-acid deletion (Arg, Ser, Gly) in PCV PK-15. Variable region 2 is in the middle of the protein (amino acids 180 to 198), and variable region 3 (amino acids 281 to 314) is at the 3' end and has 1 amino acid deleted in the predicted protein of PCV ISU-31 ORF1. ORF1 is predicted to encode a 36-kDa protein and possibly represents the major structural protein of PCV (23). It also possesses homology with putative replication-associated proteins of plant circoviruses, similar to that detected previously for PCV PK-15 (20, 23). All three domains predicted by Koonin and Ilyina (19) for Rep proteins were detected, as well as the nucleotide binding site GPPGCGKS (Fig. 2A). Three potential N-glycosylation sites were detected in the ISU-31 ORF1, compared to one site in ORF1 of PCV PK-15. The major ORF2 of the PCV ISU-31 genome is 699 bp long and is predicted to encode a 233-amino-acid protein of 28 kDa, which is similar in size to ORF2 of PCV PK-15. ORF2 was more variable than ORF1, with DNA and amino acid homologies of 67 and 65%, respectively (Fig. 2B). Comparison of the genomes of PCV ISU-31 and chicken anemia virus did not reveal any homology between these two viruses at the DNA or protein level.
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In situ hybridization. For in situ hybridization, we used tissues from pigs with PMWS which were positive for PCV by PCR. In situ hybridization was performed with sense and antisense RNA probes specific for ORF1 of PCV ISU-31.
In situ hybridization with the antisense RNA probe demonstrated PCV nucleic acid as a dark purple reaction product in the nucleus, cytoplasm, or both of numerous cells in tissues of pigs with PMWS (Fig. 4B). With the sense RNA probe, signal was present only in the nucleus of the infected cells (Fig. 4C). All negative control slides lacked reaction product (Fig. 4A).
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Isolation of virus. We also attempted isolation of PCV in PK-15 cells. Pooled tissue homogenates of lungs, lymph nodes, and liver of one animal were used for inoculation of PCV-free PK-15 cells. No signs of cytopathic effect were observed. Virus replication was monitored by PCR. All flasks of PK-15 cells inoculated with tissue homogenates were positive by PCR. IIFA was also performed on infected PK-15 cells. Results of IIFA correlated with the PCR data and are shown in Fig. 4D and E. The propagated virus (passage 4) was sequenced and found to be identical to the virus amplified from the original inoculum. We also used primers PCV75 (5'-GGGTGTTCACGCTGAATAATCCTTCCG-3') and PCV1073 (5'-CCAGGACTACAATATCCGTGTAACC-3') to amplify a 1,013-bp fragment of PCV genome from tissues of four other pigs diagnosed with PMWS from the case described above. All tissues were PCR positive (Fig. 4F), and sequencing data revealed 100% homology with the genome of PCV ISU-31.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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PMWS appears to be an emerging disease problem in North American and European swine herds (6, 14, 27, 30). Morbidity is reported to range from 5 to 50% in affected herds, and the case fatality rate approaches 100%. Although PCV infection has been associated with PMWS, the role of PCV infection in the pathogenesis of PMWS is uncertain, particularly since the PCV strain isolated from PK-15 cells is reported to be nonpathogenic (2).
In this study, PCV was detected by PCR and in situ hybridization in tissues of all six pigs exhibiting lesions consistent with PMWS. In two of these pigs, PRRSV antigen was demonstrated by immunohistochemistry. PRRSV is the causative agent of PRRS and is widespread in North America and Europe (1). PRRSV is a single-stranded positive sense RNA virus which belongs to the Arteriviridae family and causes respiratory disease and reproductive failure in pigs (24-26). PRRSV can be easily detected and differentiated from PCV by PCR, in situ hybridization, immunohistochemistry, and IIFA (5, 9, 10, 16, 21). Although both PRRS and PMWS are characterized by lymphohistiocytic interstitial pneumonia, lesions present in lymphoid tissues in the pigs in this study were characteristic of PMWS. Whereas PRRSV induces marked follicular hyperplasia of lymphoid tissues (11, 12), depletion of lymphoid tissues and replacement by macrophages and multinucleate giant cells is the hallmark of PMWS (6, 14); this lesion was consistently found in the pigs with PMWS in this study and was the major feature upon which the diagnosis of PMWS was based.
PCV was isolated from tissues of one animal and genetically characterized. Genome comparisons showed significant differences between this strain, designated PCV ISU-31, and the previously characterized PCV strain isolated as a contaminant of the porcine continuous cell line PK-15. Overall nucleotide sequence homology was 76%. ORF1 was the most conserved, with 83% nucleic acid identity and 86% amino acid identity. ORF2 was more variable, with a nucleic acid identity of 67% and amino acid identity of 65%. The patterns of small ORFs also were significantly different. Whether these genetic changes are reflected in phenotypic differences such as biological properties is not known at this time.
Because pPSP.PCV1, used for in situ hybridization, contains an insert of the PCV genome, including 36 nucleotides of noncoding region and 494 nucleotides from the 5' end of ORF1, the antisense RNA probe was expected to detect positive-strand viral DNA and double-stranded replicative-form DNA in the nucleus of the PCV-infected cells and mRNA of ORF1 in the cytoplasm of the PCV-infected cells. The sense strand RNA probe was expected to detect only the double-stranded replicative-form DNA. The nuclear staining resulting from hybridization with the sense strand RNA probe in this study suggests that the replicative intermediate form of PCV is present primarily in the nucleus of infected cells. Comparisons of consecutive slides hybridized with sense and antisense probes showed that the number of positive cells detected with the antisense strand probe was three to four times higher than that detected with the sense strand probe, making the antisense probe a much better choice for the detection of PCV in clinical samples. Many positive cells with only cytoplasmic staining were detected by the antisense probe, suggesting a significant accumulation of ORF1 mRNA and/or genomic DNA during viral replication.
The infected cells included macrophages, small mononuclear cells consistent with lymphocytes, endothelial cells, enterocytes, and pancreatic acinar epithelial cells. This is in contrast to PCV PK-15, which caused infection only in cells of mononuclear phagocyte lineage (2, 3, 22). PCV PK-15 has been shown to be nonpathogenic in pigs (2, 35). The abundant presence of PCV ISU-31 in the tissues of pigs with PMWS suggests a potentially important role for this PCV strain in PMWS; however, additional studies are needed to understand the significance of PCV in PMWS. The PCR and in situ hybridization tests developed in this study provide molecular tools for epidemiological studies of PCV infection and should be beneficial to further studies of the role of PCV in PMWS.
The common occurrence of PCV infection in swine, the demonstration of PCV-specific antibodies in humans (32), the genetic diversity among PCV strains, and the ability of PCV to cause persistent infection at least in vitro suggest the need for risk assessment of PCV as a potential zoonotic agent, considering the immense interest in the use of pigs in xenotransplantation. The PCR and in situ hybridization tests described here should provide the ability to screen pigs for PCV infection in order to provide safer donors for xenotransplantation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Veterinary Medical Research Institute, College of Veterinary Medicine, Iowa State University, Ames, IA 50011. Phone: (515) 294-0913. Fax: (515) 294-1401. E-mail: pspaul{at}iastate.edu.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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