Journal of Veterinary Diagnostic
`Investigation
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`Isolation of Porcine Circovirus-like Viruses from Pigs with a Wasting Disease in the USA and Europe
`G. M. Allan, F. McNeilly, S. Kennedy, B. Daft, J. A. Ellis, D. M. Haines, B. M. Meehan and B. M. Adair
` 1998 10: 3
`J VET Diagn Invest
`DOI: 10.1177/104063879801000102
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`- Jan 1, 1998
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`J Vet Diagn Invest 10:3–10 (1998)
`
`Isolation of porcine circovirus-like viruses from pigs with a
`wasting disease in the USA and Europe
`G. M. Allan, F. McNeilly, S. Kennedy, B. Daft, E. G. Clarke, J. A. Ellis, D. M. Haines,
`B. M. Meehan, B. M. Adair
`
`Abstract. Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning
`multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly
`diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or
`virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were
`used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the
`presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron
`microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar
`epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and
`various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen
`in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney,
`and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from
`France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from
`diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluores-
`cence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had
`variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like
`nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic
`examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No
`other virus particles were observed. Although genomic analysis for the definitive identification of these viral
`isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related
`to, although antigenically distinct from, the original PCV cell culture contaminant.
`
`Porcine circovirus (PCV) was first detected as a
`contaminant of a continuous pig kidney cell line (PK/
`15).17 Since this initial identification, this virus has
`been shown to contain a single-stranded circular DNA
`genome of 1.76 kb13,15 and is now classified in a new
`virus family, the Circoviridae.11 Serum antibody to
`PCV has been demonstrated in pigs from Germany,15,16
`Canada,7 New Zealand,10 Great Britain,8 and Northern
`Ireland.4 Experimental infections of pigs with PCV in-
`ocula, derived from contaminated PK/15 cell cultures,
`have failed to produce clinical disease.3,16 Although
`several PCV isolates have been recovered from still-
`born piglets in Northern Ireland,3 the potential of these
`viruses to cause disease has not been investigated.
`Postweaning multisystemic wasting syndrome
`(PMWS) in pigs was first identified in western Canada
`
`From the Department of Agriculture for Northern Ireland, Veter-
`inary Sciences Division, Stoney Road, Belfast BT4 3SD, Northern
`Ireland (Allan, McNeilly, Kennedy, Meehan, Adair), University of
`California–Davis School of Veterinary Medicine, San Bernardino
`Branch, 105 W. Central Avenue, San Bernadino, CA 92412 (Daft),
`and Department of Veterinary Microbiology, Western College of
`Veterinary Medicine, University of Saskatchewan, Saskatoon, SK
`S7N 5B4, Canada (Clark, Ellis, Haines).
`Received for publication October 22, 1997.
`
`in 1991.5 It is characterized by progressive weight loss,
`respiratory signs, and jaundice. Gross and histologic
`lesions include granulomatous interstitial pneumonia,
`lymphadenopathy, and lymphocytic, granulomatous
`hepatitis and nephritis. Similar syndromes have been
`described in pigs from California,6 Brittany (France),1
`and Spain (M. Domingo, personal communication).
`Porcine circovirus antigen and nucleic acid have been
`demonstrated in tissues of pigs with PMWS in Cana-
`da9 and in tissues of a pig with a similar syndrome in
`California.6 The demonstration of PCV antigen and
`nucleic acid, closely associated with lesions in a range
`of tissues from diseased pigs, has led to speculation
`that a new or modified virulent PCV may have
`emerged in the swine population of several countries.
`In this communication, we report the isolation of a
`PCV-like virus from tissues of pigs with a wasting
`disease in California and Brittany and the results of
`studies of the reactivity of these viruses against poly-
`clonal antisera and a panel of monoclonal antibodies
`raised against the PCV derived from contaminated
`PK/15 cells.17
`
`Materials and methods
`Tissue samples. Samples of lung, liver, kidney, pancreas,
`spleen, and lymph node collected from diseased pigs in Cal-
`
`3
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`4
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`Allan et al.
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`ifornia and mesenteric lymph nodes (MLN) from diseased
`pigs in Brittany were examined by in situ hybridization
`(ISH) and/or virus isolation techniques. Tissue samples were
`obtained from pigs that had clinical signs typical of PMWS,
`including progressive weight loss, respiratory disease, en-
`larged lymph nodes, and jaundice.1,5,6 For virus isolation,
`lung tissue from Californian pigs and MLN tissue from
`French pigs were frozen at ⫺70 C immediately after nec-
`ropsy and assessed for suitability for virus recovery follow-
`ing examination by ISH. For histopathologic and ISH ex-
`amination, tissue samples were fixed in 10% neutral buffered
`formalin and embedded in paraffin using standard tech-
`niques. Only tissue samples that were identified as contain-
`ing abundant PCV nucleic acid were processed for virus iso-
`lation studies.
`In situ hybridization. ISH was carried out on formalin-
`fixed tissues from diseased pigs and on formalin-fixed cov-
`erslip cell culture preparations that had been inoculated for
`virus isolation.
`Whole genomic probes for PCV and chicken anemia virus
`(CAV) were used. The bacterial plasmid pPCV 1, containing
`the replicative form (RF) of the PCV genome cloned as a
`single 1.7-kb Pst1 insert13 was used as a source of PCV-
`specific viral DNA. An analogous bacterial plasmid, pCAA
`1, containing the 2.3-kb RF of the avian circovirus CAV was
`used as a negative control.18 Glycerol stocks of both these
`plasmids were used for the production of plasmid prepara-
`tions by the alkaline lysis method14 for subsequent use as
`template DNA for probe generation.
`Circovirus probes representative of the complete genomes
`of both PCV and CAV were produced from target bacterial
`plasmid preparations (1 ␮g each) and random hexanucleotide
`primers using a commercial kita in accordance with the man-
`ufacturer’s instructions. The randomly primed digoxigenin-
`labeled probes were resuspended in a final volume of 50–
`100 ␮l sterile water prior to subsequent use for ISH.
`Formalin-fixed, paraffin-embedded tissue samples from
`diseased pigs and formalin-fixed cell culture preparations
`from cell cultures inoculated for virus isolation studies were
`processed for identification of PCV nucleic acid using the
`following protocols. Five-micrometer sections were cut from
`each tissue block, dewaxed, and rehydrated through graded
`alcohols. The tissue sections and the formalin-fixed cell cul-
`ture preparations were incubated for 15 min and 5 min, re-
`spectively, at 37 C in a 0.5% solution of proteinase Kb in
`0.05 M Tris-HCl, 5 mM ethelenediaminetetraacetic acid (pH
`7.6). The slides were then placed in 1% glycine, in auto-
`claved distilled water, for 30 sec and given 2 5-min washes
`in 0.01 M phosphate-buffered saline (PBS; pH 7.2) followed
`by 1 5-min wash in sterile distilled water. Slides were then
`air dried, and the ISH probes were applied. Each probe/tissue
`preparation was covered with a clean, degreased coverslip,
`placed in a 90 C oven for 10 min, removed, placed on an
`ice block for 1 min, and then incubated for a further 18 hr
`at 37 C. Preparations were then immersed briefly in 2⫻ sa-
`line sodium citrate buffer (SSC; pH 7.0) to remove the cov-
`erslips and given 2 5-min washes in 2⫻ SSC buffer followed
`by 2 5-min washes in PBS. Following these washes, the
`preparations were immersed in maleic buffer (0.1 M maleic
`acid, 0.15 M NaCl, pH 7.5) for 10 min and then incubated
`
`in 1% blocking reagenta in maleic buffer (blocking buffer)
`for 20 min at 37 C. The preparations were then incubated
`with a 1/250 dilution of a murine monoclonal antibody to
`digoxigenin,a diluted in blocking buffer, for 1 hr at 37 C,
`washed in PBS, and then incubated with a biotinylated anti-
`mouse IgGc at 37 C for 30 min. Preparations were again
`washed in PBS, and endogenous peroxidase activity was
`blocked by treatment with a solution of 0.5% H2O2 in PBS
`for 20 min at room temperature. The preparations were
`washed in PBS and incubated with streptavidin–peroxidase
`conjugatec for 15 min at room temperature, washed again in
`PBS, and treated with a freshly made 3-amino-9-diethylcar-
`bazole substratec for a further 5 min. Following a wash in
`running tap water, the preparations were counterstained with
`hematoxylin, ‘‘blued’’ in running tap water, and mounted
`with coverslips using an aqueous mountant (GVA Mount).c
`Controls included the use of an inappropriate probe (CAV)
`on tissues from diseased pigs and a positive probe (PCV) on
`PCV-infected cell culture preparations and tissue sections
`from nondiseased pigs.
`Virus isolation. For virus isolation, approximate 15% sus-
`pensions of tissue samples were made in minimal essential
`medium containing Earle’s salts and penicillin (100 IU/ml)
`and streptomycin (100 ␮g/ml) (MEM-SA) by grinding the
`tissue samples in a sterile pestle and mortar with sterile sand.
`This ground preparation was then suspended in the MEM-
`SA and centrifuged at 3,000 ⫻ g for 30 min at 4 C, and the
`supernatant was removed. Prior to inoculation into cell cul-
`tures, 100 ␮l of chloroform was added to 2 ml of each su-
`pernatant and mixed continuously for 10 min at room tem-
`perature. The mixture was then transferred to a microcentri-
`fuge tube and centrifuged at 3,000 ⫻ g for 10 min, and the
`supernatant was removed. This supernatant was used as an
`inoculum for virus isolation studies.
`All virus isolation studies were carried out in PK/15 cell
`cultures, previously shown to be free of contaminating PCV,3
`pestivirus, porcine adenovirus, and porcine parvovirus.
`Virus isolation studies were carried out using the follow-
`ing protocol. PK/15 cells were removed from confluent cul-
`tures using trypsin–versene and resuspended in MEM-SA
`containing 15% pestivirus-free fetal bovine serum (MEM-G)
`at a concentration of 4 ⫻ 105 cells/ml. Ten-milliliter aliquots
`of this cell suspension were mixed with 2-ml aliquots of the
`inocula described above, and the resulting mixtures were ali-
`quoted in 6-ml volumes into 2 25-ml tissue culture flasks.
`These cultures were then incubated at 37 C for 18 hr in a
`10% CO2 atmosphere. The resulting semiconfluent monolay-
`ers were treated with 300 mM D-glucosamine2 and then in-
`cubated for a further 48–72 hr at 37 C. Following this in-
`cubation, 1 of the inoculated flasks for each inoculum was
`frozen and thawed 3 times. The remaining flask for each
`inoculum was passaged by detachment of the PK/15 cells
`using trypsin–versene, resuspension of these detached cells
`in 20 ml of MEM-G, and seeding to fresh 75-ml tissue cul-
`ture flasks at a concentration of 4 ⫻ 105 cells/ml. The freshly
`seeded flasks were then ‘‘superinfected’’ by adding 5 ml of
`the appropriate cell lysate obtained after the freeze/thaw cy-
`cles. A 5-ml volume of the superinfected suspension was
`removed and seeded to a 55-mm-diameter Petri dish con-
`taining degreased sterile coverslips. The flask and coverslip
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`PCV-like viruses in diseased pigs
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`5
`
`cultures were then incubated at 37 C and processed with
`glucosamine as described above. Coverslip cell culture prep-
`arations were removed 24–48 hr after glucosamine treatment
`and fixed in acetone for 10 min at room temperature or in
`10% neutral buffered formalin for 4 hr. Following fixation,
`all coverslip preparations were stored at ⫺70 C over silica
`gel prior to use in immunocytochemical and ISH studies.
`Antibodies. Initial screening of all acetone-fixed cell cul-
`ture preparations was carried out by indirect immunofluores-
`ence (IIF) using a 1/100 dilution of pooled adult pig sera.
`This serum pool comprised sera from 25 adult sows from
`Northern Ireland and was known to contain antibodies to a
`range of porcine viruses, including PCV, porcine parvovirus,
`porcine adenovirus, and porcine reproductive and respiratory
`syndrome virus (PRRSV). The IIF procedure was performed
`by application of the diluted serum in PBS to cell culture
`preparations for 1 hr at 37 C, followed by 2 washes in PBS.
`The cell cultures were then immunostained with a 1/80 di-
`lution of a fluorescein isothiocyanate (FITC)-conjugated rab-
`bit anti-pig immunoglobulind in PBS for 1 hr, washed in
`PBS, and mounted in buffered glycerol saline prior to view-
`ing under incident UV illumination.
`FITC-conjugated hyperimmune rabbit antisera to porcine
`parvovirus, porcine adenovirus, PRRSV, classical swine fe-
`ver virus, and transmissible gastroenteritis virus (TGEV)
`were used to screen for evidence of these viral infections in
`cell culture preparations that reacted with the pooled adult
`pig serum. These conjugates were prepared and used as de-
`scribed previously.12
`All cell culture preparations reactive with the pig serum
`pool were processed by IIF with aliquots of serial 2-fold
`dilutions of a hyperimmune rabbit antiserum to PCV (PCV-
`T)e from 1/100 to 1/6,400 dilution. This serum had an IIF
`titer of 1/10,000 against acetone-fixed PK/15 cells persis-
`tently infected with the cell culture contaminant of PCV.17
`In addition, cell culture preparations reactive against the por-
`cine serum pool were processed by IIF at the same dilutions
`with a hyperimmune rabbit antiserum prepared against a cell
`culture isolate of a PCV-like virus from diseased pigs in
`Canada9 (PCV-C). This antiserum was also tested by IIF
`against the PK/15 cell culture PCV isolate at the same di-
`lutions.
`A panel of monoclonal antibodies (F99) against the cell
`culture contaminant PCV2 was reacted by IIF against ace-
`tone-fixed cell culture preparations inoculated with tissue
`preparations from French and Californian pigs and against
`the pooled polyclonal pig sera and the rabbit hyperimmune
`antiserum to the Canadian PCV-like virus. These monoclonal
`antibodies were diluted to 1/100, 1/1,000, and 1/10,000 and
`were reacted against the homologous PK/15 PCV virus as
`controls.2
`Electron microscopy. Transmission electron microscopic
`examination of selected immunopositive cell cultures was
`performed using standard techniques on cell culture prepa-
`rations that had been inoculated with suspect PCV material
`and harvested 5 days after inoculation. Cell culture super-
`natant was discarded, and the remaining cells were scraped
`into 5-ml of distilled water. This cell suspension was then
`frozen and thawed at ⫺70 C (3 cycles) and sonicated in an
`ice bath, and cell debris was removed by centrifuging the
`
`Figure 1. Lung tissue from Californian pig with wasting syn-
`drome following hybridization with a digoxigenin-labeled whole ge-
`nomic PCV probe. Note abundant hybridization signal in nuclei (ar-
`rowheads) and cytoplasm (arrows) of alveolar cells.
`
`lysate at 2,000 ⫻ g for 30 min, after which, 2 ml of the
`supernatant was transferred to a micocentrifuge tube and
`centrifuged at 13,000 ⫻ g for 1.5 hr. The supernatant was
`discarded, and the pellet was resuspended in 25 ␮l of sterile
`distilled water. This suspension was applied to a carbon-
`coated electron microscope grid for 5 min, negatively stained
`with ammonium molybdate for 30 sec, and examined in an
`electron microscope.
`
`Results
`Histopathologic findings. Severe lesions of bron-
`chointerstitial pneumonia were apparent in lung tissue
`from the Californian pigs. These lesions included fill-
`ing of bronchi and bronchioles with macrophages and
`edema fluid and dense mononuclear cell infiltration of
`alveolar septa and peribronchial, peribronchiolar, and
`interlobular connective tissue. Proliferation of type II
`pneumocytes was also apparent. Basophilic nuclear
`and cytoplasmic inclusions were seen in many cells in
`alveolar lumina and septa, bronchial epithelium, and
`peribronchial cells.
`Large pale areas were apparent throughout the cor-
`tex and paracortex of MLN of the French pigs. Nu-
`merous syncytia that contained approximately 3–20
`vesicular nuclei and infiltrated histiocytic cells were
`seen in these areas. There was infiltration of connec-
`tive tissue trabeculae with mononuclear cells, and cap-
`illary and arteriolar endothelial cells were swollen.
`Unequivocal inclusions were not seen in lymph node
`tissue from these animals.
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`6
`
`Allan et al.
`
`and peribronchiolar cells in lung tissue of the Califor-
`nian pigs (Fig. 1). Nucleic acid was also seen in pneu-
`mocytes, bronchial and bronchiolar epithelial cells,
`and endothelial cells of small arterioles, venules, and
`lymphatics. There was intense cytoplasmic and nuclear
`labeling of PCV-like nucleic acid in pancreatic acinar
`cells (Fig. 2), renal tubular epithelial cells, hepato-
`cytes, Kuppfer cells, and mononuclear cells scattered
`throughout liver, kidney, and pancreas tissue.
`PCV-like nucleic acid was detected in the cytoplasm
`of many follicular lymphocytes and intrasinusoidal
`mononuclear cells in lymph nodes of the Californian
`and French pigs. Nucleic acid was also detected in
`occasional syncytia. On the basis of ISH results, lung
`tissue from diseased Californian pigs and MLN from
`French pigs were chosen for virus isolation studies.
`Virus isolation. Cytopathic effect was not observed
`in cell cultures inoculated with specimens from the
`Californian or French pigs that had PMWS. However,
`immunostaining of acetone-fixed preparations from in-
`oculated cell cultures with the pool of polyclonal pig
`serum revealed nuclear fluorescence in many cells in
`cell cultures inoculated with lung tissue preparations
`from the Californian pigs (Fig. 3) and MLN tissue
`preparations from the French pigs. Immunostaining
`was not observed following application of FITC-con-
`jugated porcine parvovirus, porcine adenovirus, clas-
`sical swine fever virus, TGEV, or PRRSV antisera to
`these cultures. IIF staining of cell cultures reactive
`with the pool of adult pig sera was observed only with
`low dilutions of the hyperimmune rabbit antibody to
`the cell culture contaminant PCV (PCV-T) (Table 1).
`This staining was confined to the nucleus of a few cells
`and was dull (Fig. 4). In contrast, IIF staining of cell
`
`Figure 2. Pancreatic tissue from Californian pig with wasting
`syndrome following hybridization with a digoxigenin-labeled whole
`genomic PCV probe. Note hybridization signal in nuclei of many
`acinar cells near necrotic focus (top).
`
`ISH. ISH, using a PCV genomic probe, revealed the
`presence of PCV-like nucleic acid in formalin-fixed
`tissues from Californian and French pigs. No reactivity
`was observed when the PCV genomic probe was ap-
`plied to tissue sections from pigs without disease or
`when the CAV probe was applied to tissues from dis-
`eased pigs. PCV nucleic acid was identified in the cy-
`toplasm and nucleus of many infiltrated mononuclear
`cells in alveolar lumina and septa and in peribronchial
`
`Figure 3.
`Immunofluorescent staining of PK/15 cell cultures inoculated with preparations of lung tissue from diseased pigs from
`California, using a pool of adult pig sera from Northern Ireland as a primary antibody. Note intense nuclear staining in some cells.
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`PCV-like viruses in diseased pigs
`
`7
`
`Table 1. Results* of indirect immunofluorescent (IIF) staining
`of PCV-like viruses with a hyperimmune antiserum to the PK/15
`contaminant circovirus (PCV-T) and a panel of monoclonal antibod-
`ies (F99) prepared against the PK/15 contaminant and a hyperim-
`mune antiserum prepared against a PCV-like virus isolated from a
`case of PMWS in Canada (PCV-C).
`
`Virus
`
`France
`USA
`PK/15
`200
`ⱖ6,400
`PCV-T antiserum
`800
`ⱖ6,400
`ⱖ6,400
`200
`PCV-C antiserum
`⬍100
`ⱖ10,000
`100
`F99 1H4
`⬍100
`⬍100
`ⱖ10,000
`F99 4B10
`⬍100
`ⱖ10,000
`100
`F99 2B7
`⬍100
`⬍100
`ⱖ10,000
`F99 2E12
`⬍100
`ⱖ10,000
`100
`F99 1C9
`⬍100
`⬍100
`ⱖ10,000
`F99 2E1
`⬍100
`ⱖ10,000
`100
`F99 1H4
`* Reciprocal of the last serum or monoclonal antibody dilution that
`resulted in a positive IIF reaction.
`
`cultures with the hyperimmune antiserum prepared
`against the PCV-like virus isolate from Canadian pigs
`(PCV-C) produced strong nuclear and cytoplasmic
`staining at high dilutions (Table 1). This antiserum
`only immunostained the cell cultures containing the
`PK/15 PCV at low dilution (Table 1).
`ISH studies on formalin-fixed cell culture prepara-
`tions from inoculated specimens using a PCV whole
`genomic probe resulted in strong positive signal in the
`nucleus of some cells in cultures inoculated with prep-
`arations of lung tissue from Californian pigs (Fig. 5)
`and MLN tissue from French pigs (Fig. 6). Reactivity
`was not observed when this probe was applied to un-
`
`infected control cell cultures or when a whole genomic
`CAV probe was applied to the inoculated cultures.
`Electron microscopy. Electron microscopic exami-
`nation of cell culture preparations in which nuclear
`staining was apparent revealed the presence of small
`aggregates of PCV-like particles (Fig. 7) that were cir-
`cular and had a diameter of 17 nm. No other virus-
`like particles were observed in these preparations nor
`were any virus particles observed in uninoculated con-
`trol cell cultures.
`Reactivity against a panel of monoclonal antibodies.
`The results of screening acetone-fixed cell culture
`preparations inoculated with tissues of pigs with
`PMWS with a panel of 7 monoclonal antibodies to the
`cell culture contaminant PCV are presented in Table
`1. Three of the monoclonal antibodies did not react at
`a 1/100 dilution with either the Californian or French
`virus-infected cell cultures. These antibodies had ho-
`mologous titers of 1/10,000. Two monoclonal antibod-
`ies were reactive with the Californian viral isolate but
`not with the French isolate, and the remaining 2 mono-
`clonal antibodies reacted with the French isolate but
`not with the Californian isolate. The staining intensity
`of this cross-reactivity was extremely weak and less
`than that observed with the highest dilution used in
`the homologous reaction.
`Discussion
`PCV is classified as a member of the virus family
`Circoviridae because it contains a circular DNA ge-
`nome. Definitive identification of the PCV-like viruses
`isolated here as circoviruses will require genomic anal-
`ysis; however,
`the evidence strongly suggests that
`
`Figure 4.
`Immunofluorescent staining of PK/15 cell cultures inoculated with preparations of lung tissue from diseased pigs from
`California, using a hyperimmune rabbit antisera to the PK/15 contaminant circovirus as a primary antibody. Note dull nuclear staining in
`some cells.
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`8
`
`Allan et al.
`
`Figure 5.
`In situ hybridization on PK/15 cell culture preparations inoculated with lung material from diseased pigs from California,
`using a whole genomic porcine circovirus probe. Note strong hybridization signal in the nuclei of some cells (arrow).
`
`these new viruses are related to the currently classified
`PCV. Electron microscopic examination of cell cul-
`tures inoculated with tissue preparations from diseased
`pigs revealed the presence of virus particles of a size
`and morphology identical to PCV.15 In addition, ISH
`studies using a cloned genomic PCV probe on for-
`malin-fixed cell culture preparations inoculated with
`tissues derived from pigs with PMWS resulted in a
`strong positive signal in the nucleus of some cells.
`This positive signal indicates a degree of genomic ho-
`mology between the new viral isolates and the PCV
`contaminant of the PK/15 cell line. However, the PCV-
`like viruses isolated in this study from diseased pigs
`are clearly antigenically different from the original
`
`PCV.15 This antigenic difference is indicated by the
`reactivity pattern to the monoclonal antibodies against
`the PK/15 virus isolate and the results obtained fol-
`lowing attempted immunostaining of the isolates with
`a hyperimmune antiserum against this virus and with
`a hyperimmune antiserum against a PCV-like virus
`isolated from Canadian pigs that had PMWS. A weak
`reaction was obtained following application of the an-
`tibody to the PK/15 virus, and no reactivity was ob-
`served following application of higher dilutions of this
`serum. This result
`indicates a degree of antigenic
`cross-reactivity between the PCV-like isolates with the
`PK/15 PCV. However, the failure of the hyperimmune
`antiserum to the PCV-like virus isolated from diseased
`
`Figure 6.
`In situ hybridization on PK/15 cell culture preparations inoculated with lymph node material from diseased pigs from France,
`using a whole genomic porcine circovirus probe. Note strong hybridization signal in nuclei of some cells (arrow).
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`PCV-like viruses in diseased pigs
`
`9
`
`Figure 7. Electron micrographs of PK/15 cell culture lysate preparations of diseased pigs. Note PCV-like particles (arrow) in both
`preparations. 150,000⫻. A. Cell cultures inoculated with lung preparations from diseased pigs from California. B. Cell cultures inoculated
`with lymph node preparations from diseased pigs from France.
`
`pigs in Canada to immunostain the PK/15 virus at high
`dilutions while reacting with the PCV-like viruses iso-
`lated from pigs from California and Brittany at a ⬎1/
`6,400 dilution suggests that antigenic homology is lim-
`ited.
`The isolation of these antigenically distinct PCV-
`like viruses from pigs with PMWS does not in itself
`confirm that they are the causal agents of the disease
`syndrome. However, the use of ISH in this study of
`diseased pigs to identify tissues that contained large
`amounts of PCV-specific nucleic acid as an indicator
`of suitability of specimens for virus isolation studies
`has also shown a close association of the presence of
`PCV nucleic acid with lesions in the pigs tested. This
`close association with lesions in diseased pigs and the
`absence of PCV antigen and/or nucleic acid in non-
`diseased tissue has also been reported in pigs with
`PMWS in Canada9 and could indicate a strong causal
`relationship between the new PCV-like viruses and
`PMWS. Although a wasting disease in pigs, similar to
`that seen in Canada, USA, France, or Spain, has not
`been identified in Northern Ireland, the pool of adult
`pig serum used in this study to screen all inoculated
`cell cultures for the presence of PCV reacted strongly
`with the 2 new PCV-like virus isolates from pigs with
`PMWS. Following this reaction, 50 individual pig sera
`assayed by IIF at a 1/100 dilution for antibody to the
`new PCV-like viruses were positive (data not shown).
`The presence of antibody to these viruses in Northern
`Ireland, as determined by IIF, in the absence of clinical
`disease provides evidence that infection of pigs with
`PCV may not be the only factor required for devel-
`opment of PMWS. This finding may indicate infection
`of pigs in Northern Ireland with a serologically related
`but less pathogenic PCV-like virus. Further detailed
`studies using ISH and immunocytochemistry are cur-
`
`rently in progress to help elucidate the role of PCV in
`the pathogenesis of PMWS.
`
`Acknowledgements
`We are grateful to Cliff Mason for production of photo-
`graphs and William Curran for production of electron mi-
`crographs.
`
`Sources and manufacturers
`a. Boehringer Mannheim, Lewes, UK.
`b. Sigma–Aldrich Co., Poole, UK.
`c. Cambridge Bioscience, Cambridge, UK.
`d. Nordic Immunological Laboratories, Tilburg, The Netherlands.
`e.
`I. Tischer, Robert Koch Institute, Berlin, Germany.
`
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