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Double In Situ Hybridization for Simultaneous Detection and Differentiation of Porcine Circovirus 1 and 2 in Pigs with Postweaning Multisystemic Wasting Syndrome
The Veterinary Journal 2002, 164, 247±253 doi:10.1053/tvjl.2001.0697, available online at http://www.idealibrary.com on
Double In Situ Hybridization for Simultaneous Detection and Differentiation of Porcine Circovirus 1 and 2 in Pigs with Postweaning Multisystemic Wasting Syndrome J. KIM and C. CHAE Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea
SUMMARY Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin± biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis # 2002 Elsevier Science Ltd. All rights reserved. studies and diagnostic applications. KEYWORDS: Double labelling; in situ hybridization; porcine circovirus; postweaning multisystemic wasting syndrome. INTRODUCTION Porcine circovirus (PCV) is a small non-enveloped virus that contains a single-stranded circular DNA genome of 1.76 kb and is classified in a newly recognized virus family, the Circoviridae (Meehan et al., 1997), along with chicken anaemia virus (Todd et al., 1990), beak-and-feather disease virus of psittacine bird (Ritchie et al., 1989), and several plant viruses (Rohde et al., 1990; Harding et al., 1993; Boevink et al., 1995). No recognized DNA sequence homologies or common antigenic determinants exist between PCV and other currently recognized circoviruses (Morozov et al., 1998). Two types of PCV have been characterized and subsequently designated PCV1 and PCV2 (Meehan et al., 1998). PCV1 is a persistent contaminant of the
Correspondence to: Chanhee Chae, Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea. E-mail: [email protected]
continuous porcine kidney cell line, PK-15 (Tischer et al., 1974). PCV2, which differs markedly from PCV1, is commonly found in pigs with the postweaning multisystemic wasting syndrome (PMWS) (Allan et al., 1998; Meehan et al., 1998; Morozov et al., 1998; Choi & Chae, 1999; Choi et al., 2000; Kim & Chae, 2001a). However, PCV1-like virus has been isolated from pigs with the wasting disease in France (LeCann et al., 1997). Mixed infections comprising both PCV1 and PCV2 have also been reported (LeCann et al., 1998; Larochelle et al., 1999; Ouardani et al., 1999). Development of diagnostic tools that can detect and differentiate between both types of PCV simultaneously would be of significant importance in epidemiological survey programmes. Multiplex polymerase chain reaction (PCR) has been used for the detection of and differentiation between PCV1 and PCV2 (Larochelle et al., 1999; Ouardani et al., 1999; Kim et al., 2001). However, PCR allows neither localization nor identification of the PCVs in infected tissues or cells. In contrast to PCR, in situ hybridization provides cellular detail and
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histological architecture so that expression of PCV and lesions may be studied in the same section. Reported in situ hybridization techniques can detect common PCV only, without discriminating between the two porcine viruses (Allan et al., 1998; Balasch et al., 1999; Choi & Chae, 1999). Because of the disadvantages, double in situ hybridization needs to be developed for the detection and differentiation of two PCVs in formalin-fixed, paraffin-embedded tissues. The objective of the present study was to develop a double-labelling in situ hybridization to detect and differentiate simultaneously between PCV1 and PCV2 DNA in formalin-fixed, paraffin-embedded tissues from pigs with PMWS. MATERIALS AND METHODS
Samples
Formalin-fixed, paraffin-embedded tissues from 20 pigs with PMWS were used (Kim & Chae, 2001b). Cases were selected on the basis of clinical signs, histopathological lesions, and virus isolation. Tissue blocks from lymph node and spleen were used to differentiate both types of PCV using doublelabelling in situ hybridization. Negative tissue controls were from 1-day-old colostrum-deprived pigs not exposed to any types of PCV and from 7-day-old pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV).
Preparation of labelled probes
A 349-base pair (bp) region DNA fragment from open reading frame (ORF) 1 was used as a PCV1 probe. The forward and reverse primers were 5 0 -TTGCTGAGCCTAGCGACACC-3 0 (nucleotides 1369 to 1388) and 5 0 -TCCACTGCTTCAAATCGGCC-3 0 (nucleotides 1717 to 1698), respectively (Larochelle et al., 1999). For the PCV2 probe, a 481-bp region DNA fragment from ORF 2 was used. The forward and reverse primers were 5 0 -CGGATATTGTAGTCCTGGTCG-3 0 (nucleotides 1095 to 1115) and 5 0 -ACTGTCAAGGCTACCACAGTCA-3 0 (nucleotides 1570 to 1549), respectively (Ellis et al., 1999). The PCR was carried out as previously described (Ellis et al., 1999; Larochelle et al., 1999). The PCR products from each of PCV1 and PCV2 were purified with Wizard PCR Preps (Promega Biotech). The purified products were labelled by random priming with digoxigenin (DIG)-dUTP for PCV1 and by random priming with biotin-dUTP for PCV2, respectively, using a commercial kit (Boehringer Mannheim).
Porcine circovirus-infected PK-15 cell cultures
PCV1- and PCV2-infected PK-15 cell cultures were grown in two-well chamber slides as previously described (Kim et al., 1999) and used as the starting material for confirmation of specificity for two probes, PCV1 and PCV2. After 5 days of incubation at 37 C, the medium was discarded, wells were rinsed with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and the cells were fixed by PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in PBS) for 5 min at room temperature. After fixation, the chambers were removed, and the wells were rinsed for 5 min twice with PBS and dehydrated through an ethanol series (70, 80, 100%; 5 min each concentration). The dehydrated slides were stored at 4 C until use. Controls included PRRSV-infected MARC-145 cells and noninfected PCV-free PK-15 cells.
Double in situ hybridization
Four serial sections were prepared from each of the formalin-fixed, paraffin-embedded tissues. One was processed for single PCV1 in situ hybridization, one for single PCV2 in situ hybridization, one by the double in situ hybridization for both PCV1 and PCV2, and one by the double in situ hybridization for both PCV1 and PCV2 with DNase I treatment. Double in situ hybridization was performed sequentially by PCV1 followed by PCV2. Tissue sections 4mm thick were cut, placed on Superfrost/plus slides (Fisher Scientific), and stored at room temperature until processed. Just before use, sections were deparaffinized in xylene and rehydrated in phosphate-buffered saline (PBS; pH 7.4, 0.01 M) for 5 min. De-proteinization was carried out in 0.2 N HCl for 20 min at room temperature. Tissues were then digested at 37 C for 20 min in proteinase K (300mg/mL) in PBS. For each tissue examined, one section was treated with DNase I (Boehringer Mannheim) at 0.1 unit/mL in 10 mM Tris-HCl (pH 7.4) for 30 min at 37 C to remove target DNA, as a specificity control. Following digestion, tissues were fixed in paraformaldehyde 4% in PBS for 10 min. After rinsing with PBS twice, the slides were acetylated in 300 mL of 0.1 mM triethanolamine-HCl buffer (pH 8.0) to which 0.75 mL of acetic anhydride (0.25%) had been added. After 5 min, a further 0.75 mL of acetic anhydride was added, and 5 min later the slides were rinsed in 2x saline sodium citrate (SSC; 1x SSC contains 50 mM NaCl and 15 mM sodium citrate, pH 7.0). Hybridization was carried out overnight at 45 C. The digoxigenin-labelled PCV1 (1 ng/mL) and
DOUBLE IN SITU HYBRIDIZATION FOR PCV-1 AND PCV-2
biotinylated PCV2 probe (1 ng/mL), respectively, was diluted in 50 mL of standard hybridization buffer that consisted of 2x SSC containing deionized formamide 50%, salmon sperm DNA (Oncor) 10 mg, sodium dodecyl sulphate (SDS) 0.02%, and dextran sulphate solution (50% concentration in PBS) 50%; it was then heated for 10 min in a 95 C heating block, and quenched on ice. Each of two probes was layered over the tissue section. Fluid was held in place by a coverslip, the edges of which were sealed with rubber cement, heated for 10 min in a 95 C heating block, and then quenched on ice. After overnight hybridization, sections were thoroughly washed, twice in 4x SSC for 5 min at room temperature, twice in 2x SSC for 5 min at 40 C, twice in 2x SSC for 5 min at room temperature, twice in 0.2x SSC for 5 min at 40 C, twice in 0.2x SSC for 5 min, once in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 5 min and once in 1x blocking reagent (Boehringer Mannheim) for 40 min at room temperature. For detection of PCV1 hybridization, sections were incubated 1 h with anti-digoxigenin conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1 : 300 in 1x blocking reagent (Boehringer Mannheim). After three washes in 0.1 mM triethanolamine-HCl buffer (pH 8.0), the final reaction was produced by immersing the sections in a solution of Vector red substrate (Vector Laboratories) for 20 min at room temperature. Inhibition of residual alkaline phosphatase was carried out immediately after detection of PCV1 DNA. The slides were washed in 0.1 mM triethanolamine-HCl buffer (pH 8.0) for 10 min at room temperature. After incubation with Trisethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HC1 and 1 mM EDTA, pH 8.0) for 10 min at 85 C, sections were thoroughly washed in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 5 min to remove the EDTA. Then, tissue sections were subjected to the visualization procedure of PCV2.
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For detection of PCV2 hybridization, sections were incubated 1 h with streptavidin conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1 : 500 in 1x blocking reagent (Boehringer Mannheim). After three washes in 0.1 mM triethanolamine-HCl buffer (pH 8.0), substrate consisting of nitroblue tetrazolium (NBT) and 5-bromocresy1-3-indolylphosphate (BCIP) was layered over the sections. Colour was allowed to develop for 1±4 h in the dark. Substrate development was stopped by dipping slides briefly in tri-ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Sections were counterstained with 0.5% methyl green. The slides were then washed with distilled water for one min and dried completely.
Virus isolation
Specimens from 20 pigs with PMWS were processed to attempt virus isolation as previously described (Ouardani et al., 1999). Lymph nodes and spleens from each pig were pooled for virus isolation. PCV-free PK-15 cells, kindly provided by Dr. Keith West of Prairie Diagnostic Services in Saskatchewan, Canada, were used to isolate PCV1 and PCV2 from tissue suspensions.
RESULTS
Double in situ hybridization
All samples which were positive by single in situ hybridization were also positive by double in situ hybridization. The five samples which were positive for PCV1 and PCV2 using single in situ hybridization, were also positive for PCV1 and PCV2 using double in situ hybridization. The 15 samples positive for PCV2 only using single in situ hybridization, were also positive for PCV2 only using double in situ hybridization (Table I).
Table I Summary of single and double in situ hybridization (ISH), and virus isolation results on samples from pigs with postweaning multisystemic wasting syndrome Number of pigs
Single ISH
Double ISH
Virus isolation
PCV1
PCV2
PCV1
PCV2
PCV1
PCV2
5 6 9
ÿ ÿ
ÿ ÿ
ÿ ÿ
ÿ
Total
5
20
5
20
5
11
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Fig. 1. Consecutive serial sections of a lymph node from a pig naturally infected with porcine circovirus 1 and 2. (A) PCV1 DNA (red reaction product) is detected in multinucleated giant cells. Single in situ hybridization; Vector red, methyl green counterstain. (B) PCV2 DNA (dark brown reaction product) is detected in multinucleated giant cells. Single in situ hybridization; nitroblue tetrazolium/ 5-bromocresyl-3-indolylphosphate, methyl green counterstain.
The binding spectra of both PCV1 and PCV2 in the double in situ hybridization corresponded to those observed in single in situ hybridization, i.e., there was neither unwanted cross-reactivity nor a decrease in staining. The morphology of host cells was preserved despite the relatively high temperature required during parts of the incubation procedure. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. For PCV1- and PCV2-infected PK-15 cells hybridized with the DIGlabelled PCV1 and biotinylated PCV2 probes, respectively, PCV1 and PCV2 DNA signals were specific since they were completely absent from the mock-infected PK-15 cells. Signal intensity varied within and between histological structures in any one section and between pigs. Both PCV1 and PCV2 DNA were detected lymph nodes and spleens of PMWS cases. Intense specific staining for both viruses was most often seen in cells within necrotic germinal centres. Cells staining positive for both viruses included macrophage-like cells with one or two large round nuclei and abundant cytoplasm. In addition, scattered macrophages exhibited a less intense, dispersed hybridization signal in the cytoplasm. The distribution of positive cells in the lymph node was multifocal. In the spleen, PCV-positive cells, which were scattered in macrophages, had large oval nuclei and moderate cytoplasm. The distribution of positive cells in the spleen was patchy. The performance of single in situ hybridization for PCV1 (Fig. 1a) and PCV2 (Fig. 1b) respectively, on serial sections of lymph node and spleen indicated a very
Fig. 2. PCV1 DNA (red reaction product; arrow) and PCV2 DNA (dark brown reaction product; arrowhead) is detected simultaneously in a macrophage from a lymph node. Double in situ hybridization; Vector red, nitroblue tetrazolium/5-bromocresyl-3indolylphosphate, methyl green counterstain.
close cell-to-cell correlation between the signals of both techniques. Co-localization of hybridization signal for both PCV1 and PCV2 was present in
DOUBLE IN SITU HYBRIDIZATION FOR PCV-1 AND PCV-2
macrophages (Fig. 2) or multinucleated giant cells of the lymph node and spleen. No hybridization signal was consistently seen in tissue sections tested with a solution of DNase I before in situ hybridization. Sections from the two negative control pigs showed no hybridization signals for both PCV1 and PCV2. To ensure adequate suppression of residual alkaline phosphatase in the tissue sections, alkaline phosphatase activity was blocked by EDTA buffer prior to development with Vector red. Hybridization signals were not seen in these tissue sections.
Virus isolation
Table I summarized the number of pigs in which PCV1 and/or PCV2 was detected by virus isolation and double in situ hybridization. Five samples were positive for PCV1 and PCV2 by two tests and six samples were positive for PCV2 by two tests. Nine samples were positive for PCV2 by double in situ hybridization. DISCUSSION In this study we describe the development of double in situ hybridization suitable for the differentiation between PCV1 and PCV2 by formalin-fixed, paraffinembedded tissues and the distribution of two viruses in lymphoid tissues. In situ hybridization is a valuable adjunct to the standard DNA extraction techniques for evaluating gene expression in tissues and cells. Its major advantage is the ability to determine which tissues or cells in a mixed population are expressing the DNA of interest. Nine samples positive for PCV2 by in situ hybridization were negative by virus isolation, indicating that in situ hybridization is more sensitive to detect PCV2 than virus isolation. A PCV probe can specifically differentiate each type of PCV in paraformaldehyde-fixed cells in vitro but not in formalin-fixed, paraffin-embedded tissues (Nawagitgul et al., 2000). However, in that study, RNA probes were used instead of DNA probes. In situ hybridization with RNA probes is generally more sensitive than hybridization with DNA probes but requires more exacting conditions (Brown, 1998). If the latter are not met, false-negative results may occur (Brown, 1998). However, because PCV is a single-stranded DNA virus (Meehan et al., 1997), DNA probes are not only as sensitive as RNA probes but also more reliable for diagnostic purpose (Choi & Chae, 1999). In the present and in previous studies, the large amounts of PCV DNA expressed in macrophage
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strongly suggested that macrophage lineages permit PCV replication and thus represent the main target of the virus (Allan et al., 1994, 1998; Morozov et al., 1998; Choi & Chae, 1999). Our data also show that macrophages may be co-infected with both viruses. The co-localization of PCV1 and PCV2 in the same population of lymphoid tissues suggests that PCV1 and PCV2 may target the same cells in vivo. When interpreting positive in situ hybridization signals in cells of the macrophage lineage, one must consider that the cell is truly permissive to viral infection resulting in a positive signal or that the positive signal merely reflects phagocytized viral antigen/ DNA from neighbouring infected cells. Although the biological function of co-infection is not yet known, it is possible that one virus complements replication of the other, or the same physiological factors may enhance replication of both viruses. The data that no pig was infected with PCV1, only in pigs with PMWS, suggest that PCV1 is not an essential aetiology for PMWS. Further studies are required to define better the interaction between PCV1 and PCV2 in the pathogenesis of PMWS. Formalin fixation is the standard method for tissue preservation in veterinary and human medicine and this material forms the major source of tissues for many studies. Formalin-fixed tissues allow veterinary practitioners to ship tissue samples for PCV identification in a well-preserved, noninfectious state. However, formalin fixation can denature antigens, which can lead to false-negative results using immunohistochemistry (Rickert & Maliniak, 1989; Haines & Chelack, 1991). Unlike immunohistochemistry, in situ hybridization is less susceptible to structural alteration caused by fixation. Use of DNA probes and in situ hybridization eliminates possibilities for error caused by antigen cross-reactivity or by the alteration of binding sites during tissue process (Cheon & Chae, 2000). The present method offers the advantages of differential detection of two closely related viruses in one section and avoids the time-consuming comparison of serial sections. The results of our study indicate that alkaline phosphatase can be employed in double in situ hybridization techniques for simultaneous detection of PCV1 and PCV2 in formalin-fixed, paraffinembedded tissues. The use of alkaline phosphatase in double in situ hybridization should also alleviate one problem encountered when using peroxidase as the enzyme. For instance, the resultant diaminobenzidine (DAB) polymer can interfere with subsequent in situ hybridization when a peroxidase-linked DNA probe is used with DAB as the chromogen
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(Brahic et al., 1994), EDTA buffer effectively destroys residual alkaline phosphatase activity in tissue sections in the present study. Developing alkaline phosphatase activity as red and brown-black precipitates respectively, can be regarded as a successful combination because the two colours can be well discriminated from each other. We found that some double-labelling experiments needed a prolonged incubation in order to detect alkaline phosphatase activity. The order of application of the techniques used is also important. A difference in results was noted when the incubation of alkaline phosphatase technique with Vector red and alkaline phosphatase visualized by NBT/BCIP were reverse (data not shown). This is due to the different sensitivity between NBT/BCIP and Vector red. It is therefore recommended firstly to visualize the alkaline phosphatase with Vector red, followed by NBT/BCIP. The double in situ hybridization described here can be considered useful for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed paraffin-embedded tissues from pigs with PMWS. It can be applied to various types of biological materials, including tissue sections. Because of the applicability of this technique to a wide variety of materials, the double in situ hybridization technique can be regarded as a useful tool for routine diagnosis of mixed viral infections.
ACKNOWLEDGEMENTS The research reported here was supported by Ministry of Agriculture, Forestry and FisheriesSpecial Grants Research Program (MAFF-SGRP), and Brain Korea 21 Project, Republic of Korea. REFERENCES ALLAN, G. M., PHENIX, K. V., TODD, D. & MCNULTY, M. S. (1994). Some biological and physico-chemical properties of porcine circovirus. Journal of Veterinary Medicine B 41, 17±26. ALLAN, G. M., MCNEILLY, F., KENNEDY, S., DAFT, B., CLARKE, E. G., ELLIS, J. A., HAINES, D. M., MEEHAN, B. M. & ADAIR, B. M. (1998). Isolation of porcine circoviruslike viruses from pigs with a wasting disease in the USA and Europe. Journal of Veterinary Diagnostic Investigation 10, 3±10. BALASCH, M., SEGALES, J., ROSELL, C., DOMINGO, M., MANKERTZ, A., URNIZA, A. & PLANA-DURAN, J. (1999). Experimental inoculation of conventional pigs with tissue homogenates from pigs with post-weaning multisystemic wasting syndrome. Journal of Comparative Pathology 121, 139±48.
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LAROCHELLE, R., ANTAYA, M., MORIN, M. & MAGAR, R. (1999). Typing of porcine circovirus in clinical specimens by multiplex PCR. Journal of Virological Methods 80, 69±75. LECANN, P., ALBINA, E., MADEC, F., CARIOLET, R. & JESTIN, A. (1997). Piglet wasting disease. Veterinary Record 141, 660. LECANN, P., BLANCHARD, P., ARNAULD, C., ALBINA, E., HUTET, E., MADEC, F., MORVAN, P., EVENO, E., CARIOLET, R. & JESTIN, A. (1998). Identification of porcine circovirus associated with piglet wasting disease. Proceedings of the International Pig Veterinary Society Congress 15, 402. MEEHAN, B. M., CREELAN, J. L., MCNULTY M. S. & TODD, D. (1997). Sequence of porcine circovirus DNA: affinities with plant circoviruses. Journal of General Virology 78, 221±7. MEEHAN, B. M., MCNEILLY, F., TODD, D., KENNEDY, S., JEWHURST, V. A., ELLIS, J. A., HASSARD, L. E., CLARK, E. G., HAINES, D. M. & ALLAN, G. M. (1998). Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. Journal of General Virology 79, 2171±9. MOROZOV, I., SIRINARUMITR, T., SORDEN, S. D., HALBUR, P. G., MORGAN, M. K., YOON, K.-J. & PAUL, P. S. (1998). Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. Journal of Clinical Microbiology 36, 2535±41. NAWAGITGUL, P., MOROZOV, I., SIRINARUMITR, T., SORDEN, S. D. & PAUL, P. S. (2000). Development of probes to
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Double In Situ Hybridization for Simultaneous Detection and Differentiation of Porcine Circovirus 1 and 2 in Pigs with Postweaning Multisystemic Wasting Syndrome J. KIM and C. CHAE Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea
SUMMARY Double in situ hybridization using a digoxigenin-labelled porcine circovirus 1 (PCV1) and biotinylated PCV2 probe, was developed for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed, paraffin-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The combination of an alkaline phosphatase conjugated antidigoxigenin system with alkaline phosphatase conjugated streptavidin± biotin system allowed identification of PCV1 and/or PCV2. No evidence of cross-reaction was observed. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. Co-localization of hybridization signal for both PCV1 and PCV2 was present in macrophages and multinucleated giant cells of the lymph node and spleen. This double-labelling technique for the differentiation between PCV1 and PCV2 is suitable for pathogenesis # 2002 Elsevier Science Ltd. All rights reserved. studies and diagnostic applications. KEYWORDS: Double labelling; in situ hybridization; porcine circovirus; postweaning multisystemic wasting syndrome. INTRODUCTION Porcine circovirus (PCV) is a small non-enveloped virus that contains a single-stranded circular DNA genome of 1.76 kb and is classified in a newly recognized virus family, the Circoviridae (Meehan et al., 1997), along with chicken anaemia virus (Todd et al., 1990), beak-and-feather disease virus of psittacine bird (Ritchie et al., 1989), and several plant viruses (Rohde et al., 1990; Harding et al., 1993; Boevink et al., 1995). No recognized DNA sequence homologies or common antigenic determinants exist between PCV and other currently recognized circoviruses (Morozov et al., 1998). Two types of PCV have been characterized and subsequently designated PCV1 and PCV2 (Meehan et al., 1998). PCV1 is a persistent contaminant of the
Correspondence to: Chanhee Chae, Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, Suwon 441-744, Kyounggi-Do, Republic of Korea. E-mail: [email protected]
continuous porcine kidney cell line, PK-15 (Tischer et al., 1974). PCV2, which differs markedly from PCV1, is commonly found in pigs with the postweaning multisystemic wasting syndrome (PMWS) (Allan et al., 1998; Meehan et al., 1998; Morozov et al., 1998; Choi & Chae, 1999; Choi et al., 2000; Kim & Chae, 2001a). However, PCV1-like virus has been isolated from pigs with the wasting disease in France (LeCann et al., 1997). Mixed infections comprising both PCV1 and PCV2 have also been reported (LeCann et al., 1998; Larochelle et al., 1999; Ouardani et al., 1999). Development of diagnostic tools that can detect and differentiate between both types of PCV simultaneously would be of significant importance in epidemiological survey programmes. Multiplex polymerase chain reaction (PCR) has been used for the detection of and differentiation between PCV1 and PCV2 (Larochelle et al., 1999; Ouardani et al., 1999; Kim et al., 2001). However, PCR allows neither localization nor identification of the PCVs in infected tissues or cells. In contrast to PCR, in situ hybridization provides cellular detail and
1090-0233/02/$ ± see front matter # 2002 Elsevier Science Ltd. All rights reserved.
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histological architecture so that expression of PCV and lesions may be studied in the same section. Reported in situ hybridization techniques can detect common PCV only, without discriminating between the two porcine viruses (Allan et al., 1998; Balasch et al., 1999; Choi & Chae, 1999). Because of the disadvantages, double in situ hybridization needs to be developed for the detection and differentiation of two PCVs in formalin-fixed, paraffin-embedded tissues. The objective of the present study was to develop a double-labelling in situ hybridization to detect and differentiate simultaneously between PCV1 and PCV2 DNA in formalin-fixed, paraffin-embedded tissues from pigs with PMWS. MATERIALS AND METHODS
Samples
Formalin-fixed, paraffin-embedded tissues from 20 pigs with PMWS were used (Kim & Chae, 2001b). Cases were selected on the basis of clinical signs, histopathological lesions, and virus isolation. Tissue blocks from lymph node and spleen were used to differentiate both types of PCV using doublelabelling in situ hybridization. Negative tissue controls were from 1-day-old colostrum-deprived pigs not exposed to any types of PCV and from 7-day-old pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV).
Preparation of labelled probes
A 349-base pair (bp) region DNA fragment from open reading frame (ORF) 1 was used as a PCV1 probe. The forward and reverse primers were 5 0 -TTGCTGAGCCTAGCGACACC-3 0 (nucleotides 1369 to 1388) and 5 0 -TCCACTGCTTCAAATCGGCC-3 0 (nucleotides 1717 to 1698), respectively (Larochelle et al., 1999). For the PCV2 probe, a 481-bp region DNA fragment from ORF 2 was used. The forward and reverse primers were 5 0 -CGGATATTGTAGTCCTGGTCG-3 0 (nucleotides 1095 to 1115) and 5 0 -ACTGTCAAGGCTACCACAGTCA-3 0 (nucleotides 1570 to 1549), respectively (Ellis et al., 1999). The PCR was carried out as previously described (Ellis et al., 1999; Larochelle et al., 1999). The PCR products from each of PCV1 and PCV2 were purified with Wizard PCR Preps (Promega Biotech). The purified products were labelled by random priming with digoxigenin (DIG)-dUTP for PCV1 and by random priming with biotin-dUTP for PCV2, respectively, using a commercial kit (Boehringer Mannheim).
Porcine circovirus-infected PK-15 cell cultures
PCV1- and PCV2-infected PK-15 cell cultures were grown in two-well chamber slides as previously described (Kim et al., 1999) and used as the starting material for confirmation of specificity for two probes, PCV1 and PCV2. After 5 days of incubation at 37 C, the medium was discarded, wells were rinsed with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) and the cells were fixed by PLP (4% paraformaldehyde, 100 mM L-lysine dihydrochloride, 10 mM sodium m-periodate in PBS) for 5 min at room temperature. After fixation, the chambers were removed, and the wells were rinsed for 5 min twice with PBS and dehydrated through an ethanol series (70, 80, 100%; 5 min each concentration). The dehydrated slides were stored at 4 C until use. Controls included PRRSV-infected MARC-145 cells and noninfected PCV-free PK-15 cells.
Double in situ hybridization
Four serial sections were prepared from each of the formalin-fixed, paraffin-embedded tissues. One was processed for single PCV1 in situ hybridization, one for single PCV2 in situ hybridization, one by the double in situ hybridization for both PCV1 and PCV2, and one by the double in situ hybridization for both PCV1 and PCV2 with DNase I treatment. Double in situ hybridization was performed sequentially by PCV1 followed by PCV2. Tissue sections 4mm thick were cut, placed on Superfrost/plus slides (Fisher Scientific), and stored at room temperature until processed. Just before use, sections were deparaffinized in xylene and rehydrated in phosphate-buffered saline (PBS; pH 7.4, 0.01 M) for 5 min. De-proteinization was carried out in 0.2 N HCl for 20 min at room temperature. Tissues were then digested at 37 C for 20 min in proteinase K (300mg/mL) in PBS. For each tissue examined, one section was treated with DNase I (Boehringer Mannheim) at 0.1 unit/mL in 10 mM Tris-HCl (pH 7.4) for 30 min at 37 C to remove target DNA, as a specificity control. Following digestion, tissues were fixed in paraformaldehyde 4% in PBS for 10 min. After rinsing with PBS twice, the slides were acetylated in 300 mL of 0.1 mM triethanolamine-HCl buffer (pH 8.0) to which 0.75 mL of acetic anhydride (0.25%) had been added. After 5 min, a further 0.75 mL of acetic anhydride was added, and 5 min later the slides were rinsed in 2x saline sodium citrate (SSC; 1x SSC contains 50 mM NaCl and 15 mM sodium citrate, pH 7.0). Hybridization was carried out overnight at 45 C. The digoxigenin-labelled PCV1 (1 ng/mL) and
DOUBLE IN SITU HYBRIDIZATION FOR PCV-1 AND PCV-2
biotinylated PCV2 probe (1 ng/mL), respectively, was diluted in 50 mL of standard hybridization buffer that consisted of 2x SSC containing deionized formamide 50%, salmon sperm DNA (Oncor) 10 mg, sodium dodecyl sulphate (SDS) 0.02%, and dextran sulphate solution (50% concentration in PBS) 50%; it was then heated for 10 min in a 95 C heating block, and quenched on ice. Each of two probes was layered over the tissue section. Fluid was held in place by a coverslip, the edges of which were sealed with rubber cement, heated for 10 min in a 95 C heating block, and then quenched on ice. After overnight hybridization, sections were thoroughly washed, twice in 4x SSC for 5 min at room temperature, twice in 2x SSC for 5 min at 40 C, twice in 2x SSC for 5 min at room temperature, twice in 0.2x SSC for 5 min at 40 C, twice in 0.2x SSC for 5 min, once in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 5 min and once in 1x blocking reagent (Boehringer Mannheim) for 40 min at room temperature. For detection of PCV1 hybridization, sections were incubated 1 h with anti-digoxigenin conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1 : 300 in 1x blocking reagent (Boehringer Mannheim). After three washes in 0.1 mM triethanolamine-HCl buffer (pH 8.0), the final reaction was produced by immersing the sections in a solution of Vector red substrate (Vector Laboratories) for 20 min at room temperature. Inhibition of residual alkaline phosphatase was carried out immediately after detection of PCV1 DNA. The slides were washed in 0.1 mM triethanolamine-HCl buffer (pH 8.0) for 10 min at room temperature. After incubation with Trisethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HC1 and 1 mM EDTA, pH 8.0) for 10 min at 85 C, sections were thoroughly washed in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, pH 7.5) for 5 min to remove the EDTA. Then, tissue sections were subjected to the visualization procedure of PCV2.
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For detection of PCV2 hybridization, sections were incubated 1 h with streptavidin conjugated with alkaline phosphatase (Boehringer Mannheim) diluted 1 : 500 in 1x blocking reagent (Boehringer Mannheim). After three washes in 0.1 mM triethanolamine-HCl buffer (pH 8.0), substrate consisting of nitroblue tetrazolium (NBT) and 5-bromocresy1-3-indolylphosphate (BCIP) was layered over the sections. Colour was allowed to develop for 1±4 h in the dark. Substrate development was stopped by dipping slides briefly in tri-ethylenediaminetetraacetic acid (EDTA) buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). Sections were counterstained with 0.5% methyl green. The slides were then washed with distilled water for one min and dried completely.
Virus isolation
Specimens from 20 pigs with PMWS were processed to attempt virus isolation as previously described (Ouardani et al., 1999). Lymph nodes and spleens from each pig were pooled for virus isolation. PCV-free PK-15 cells, kindly provided by Dr. Keith West of Prairie Diagnostic Services in Saskatchewan, Canada, were used to isolate PCV1 and PCV2 from tissue suspensions.
RESULTS
Double in situ hybridization
All samples which were positive by single in situ hybridization were also positive by double in situ hybridization. The five samples which were positive for PCV1 and PCV2 using single in situ hybridization, were also positive for PCV1 and PCV2 using double in situ hybridization. The 15 samples positive for PCV2 only using single in situ hybridization, were also positive for PCV2 only using double in situ hybridization (Table I).
Table I Summary of single and double in situ hybridization (ISH), and virus isolation results on samples from pigs with postweaning multisystemic wasting syndrome Number of pigs
Single ISH
Double ISH
Virus isolation
PCV1
PCV2
PCV1
PCV2
PCV1
PCV2
5 6 9
ÿ ÿ
ÿ ÿ
ÿ ÿ
ÿ
Total
5
20
5
20
5
11
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Fig. 1. Consecutive serial sections of a lymph node from a pig naturally infected with porcine circovirus 1 and 2. (A) PCV1 DNA (red reaction product) is detected in multinucleated giant cells. Single in situ hybridization; Vector red, methyl green counterstain. (B) PCV2 DNA (dark brown reaction product) is detected in multinucleated giant cells. Single in situ hybridization; nitroblue tetrazolium/ 5-bromocresyl-3-indolylphosphate, methyl green counterstain.
The binding spectra of both PCV1 and PCV2 in the double in situ hybridization corresponded to those observed in single in situ hybridization, i.e., there was neither unwanted cross-reactivity nor a decrease in staining. The morphology of host cells was preserved despite the relatively high temperature required during parts of the incubation procedure. Positive cells exhibited a red or dark brown reaction product for PCV1 and PCV2, respectively. Both PCV DNAs were observed mainly in the cytoplasm but occasionally in the nucleus. For PCV1- and PCV2-infected PK-15 cells hybridized with the DIGlabelled PCV1 and biotinylated PCV2 probes, respectively, PCV1 and PCV2 DNA signals were specific since they were completely absent from the mock-infected PK-15 cells. Signal intensity varied within and between histological structures in any one section and between pigs. Both PCV1 and PCV2 DNA were detected lymph nodes and spleens of PMWS cases. Intense specific staining for both viruses was most often seen in cells within necrotic germinal centres. Cells staining positive for both viruses included macrophage-like cells with one or two large round nuclei and abundant cytoplasm. In addition, scattered macrophages exhibited a less intense, dispersed hybridization signal in the cytoplasm. The distribution of positive cells in the lymph node was multifocal. In the spleen, PCV-positive cells, which were scattered in macrophages, had large oval nuclei and moderate cytoplasm. The distribution of positive cells in the spleen was patchy. The performance of single in situ hybridization for PCV1 (Fig. 1a) and PCV2 (Fig. 1b) respectively, on serial sections of lymph node and spleen indicated a very
Fig. 2. PCV1 DNA (red reaction product; arrow) and PCV2 DNA (dark brown reaction product; arrowhead) is detected simultaneously in a macrophage from a lymph node. Double in situ hybridization; Vector red, nitroblue tetrazolium/5-bromocresyl-3indolylphosphate, methyl green counterstain.
close cell-to-cell correlation between the signals of both techniques. Co-localization of hybridization signal for both PCV1 and PCV2 was present in
DOUBLE IN SITU HYBRIDIZATION FOR PCV-1 AND PCV-2
macrophages (Fig. 2) or multinucleated giant cells of the lymph node and spleen. No hybridization signal was consistently seen in tissue sections tested with a solution of DNase I before in situ hybridization. Sections from the two negative control pigs showed no hybridization signals for both PCV1 and PCV2. To ensure adequate suppression of residual alkaline phosphatase in the tissue sections, alkaline phosphatase activity was blocked by EDTA buffer prior to development with Vector red. Hybridization signals were not seen in these tissue sections.
Virus isolation
Table I summarized the number of pigs in which PCV1 and/or PCV2 was detected by virus isolation and double in situ hybridization. Five samples were positive for PCV1 and PCV2 by two tests and six samples were positive for PCV2 by two tests. Nine samples were positive for PCV2 by double in situ hybridization. DISCUSSION In this study we describe the development of double in situ hybridization suitable for the differentiation between PCV1 and PCV2 by formalin-fixed, paraffinembedded tissues and the distribution of two viruses in lymphoid tissues. In situ hybridization is a valuable adjunct to the standard DNA extraction techniques for evaluating gene expression in tissues and cells. Its major advantage is the ability to determine which tissues or cells in a mixed population are expressing the DNA of interest. Nine samples positive for PCV2 by in situ hybridization were negative by virus isolation, indicating that in situ hybridization is more sensitive to detect PCV2 than virus isolation. A PCV probe can specifically differentiate each type of PCV in paraformaldehyde-fixed cells in vitro but not in formalin-fixed, paraffin-embedded tissues (Nawagitgul et al., 2000). However, in that study, RNA probes were used instead of DNA probes. In situ hybridization with RNA probes is generally more sensitive than hybridization with DNA probes but requires more exacting conditions (Brown, 1998). If the latter are not met, false-negative results may occur (Brown, 1998). However, because PCV is a single-stranded DNA virus (Meehan et al., 1997), DNA probes are not only as sensitive as RNA probes but also more reliable for diagnostic purpose (Choi & Chae, 1999). In the present and in previous studies, the large amounts of PCV DNA expressed in macrophage
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strongly suggested that macrophage lineages permit PCV replication and thus represent the main target of the virus (Allan et al., 1994, 1998; Morozov et al., 1998; Choi & Chae, 1999). Our data also show that macrophages may be co-infected with both viruses. The co-localization of PCV1 and PCV2 in the same population of lymphoid tissues suggests that PCV1 and PCV2 may target the same cells in vivo. When interpreting positive in situ hybridization signals in cells of the macrophage lineage, one must consider that the cell is truly permissive to viral infection resulting in a positive signal or that the positive signal merely reflects phagocytized viral antigen/ DNA from neighbouring infected cells. Although the biological function of co-infection is not yet known, it is possible that one virus complements replication of the other, or the same physiological factors may enhance replication of both viruses. The data that no pig was infected with PCV1, only in pigs with PMWS, suggest that PCV1 is not an essential aetiology for PMWS. Further studies are required to define better the interaction between PCV1 and PCV2 in the pathogenesis of PMWS. Formalin fixation is the standard method for tissue preservation in veterinary and human medicine and this material forms the major source of tissues for many studies. Formalin-fixed tissues allow veterinary practitioners to ship tissue samples for PCV identification in a well-preserved, noninfectious state. However, formalin fixation can denature antigens, which can lead to false-negative results using immunohistochemistry (Rickert & Maliniak, 1989; Haines & Chelack, 1991). Unlike immunohistochemistry, in situ hybridization is less susceptible to structural alteration caused by fixation. Use of DNA probes and in situ hybridization eliminates possibilities for error caused by antigen cross-reactivity or by the alteration of binding sites during tissue process (Cheon & Chae, 2000). The present method offers the advantages of differential detection of two closely related viruses in one section and avoids the time-consuming comparison of serial sections. The results of our study indicate that alkaline phosphatase can be employed in double in situ hybridization techniques for simultaneous detection of PCV1 and PCV2 in formalin-fixed, paraffinembedded tissues. The use of alkaline phosphatase in double in situ hybridization should also alleviate one problem encountered when using peroxidase as the enzyme. For instance, the resultant diaminobenzidine (DAB) polymer can interfere with subsequent in situ hybridization when a peroxidase-linked DNA probe is used with DAB as the chromogen
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(Brahic et al., 1994), EDTA buffer effectively destroys residual alkaline phosphatase activity in tissue sections in the present study. Developing alkaline phosphatase activity as red and brown-black precipitates respectively, can be regarded as a successful combination because the two colours can be well discriminated from each other. We found that some double-labelling experiments needed a prolonged incubation in order to detect alkaline phosphatase activity. The order of application of the techniques used is also important. A difference in results was noted when the incubation of alkaline phosphatase technique with Vector red and alkaline phosphatase visualized by NBT/BCIP were reverse (data not shown). This is due to the different sensitivity between NBT/BCIP and Vector red. It is therefore recommended firstly to visualize the alkaline phosphatase with Vector red, followed by NBT/BCIP. The double in situ hybridization described here can be considered useful for the simultaneous detection and differentiation of PCV1 and PCV2 in formalin-fixed paraffin-embedded tissues from pigs with PMWS. It can be applied to various types of biological materials, including tissue sections. Because of the applicability of this technique to a wide variety of materials, the double in situ hybridization technique can be regarded as a useful tool for routine diagnosis of mixed viral infections.
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