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Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture
Veterinary Microbiology 67 (1999) 1±12
Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture M. Dauber* Federal Research Centre for Virus Diseases of Animals, Friedrich Loeffler Institutes, Institute of Diagnostic Virology, Boddenblick 5a, D-17498 Insel Riems, Germany Received 18 September 1998; accepted 15 February 1999
Abstract A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Pig-viruses; Porcine enterovirus; Monoclonal antibody; Diagnosis; Immuno¯uorescence
1. Introduction Porcine enteroviruses (PEV) have been classified to the genus Enterovirus of the family Picornaviridae (Honda et al., 1990; Auerbach et al., 1994; Minor et al., 1995) and at least 13 species (serotypes) can be distinguished. Classification of PEV is primarily based on a minimum relationship indicated by serum neutralization titre determined in cross-reaction tests. When the heterologous titre of a PEV reaches at least 5% of the homologous one both strains are assigned to the same serotype (Dunne et al., 1971; Knowles et al., 1979). Three groups of PEV can be distinguished by parameters such as physico-chemical properties, type of cytopathic effect in pig kidney cells, cross-reactions, and genomic conformity. Most of these viruses are integrated into serotype group I * Tel.: +49-38351-70; fax: +49-38351-7219; e-mail: [email protected] 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 2 4 - 3
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(PEV1-7, 11-13), which have many characteristic features in common but differ in some points from all the other enteroviruses (Knowles, 1988). PEV-8 represents group II and serotypes 9 and 10 form group III. The virions are icosahedral particles and have a 28± 30 nm capsid without envelope, made up of 60 copies of four proteins, VP1 to VP4. The genome consists of a single stranded monopartite RNA with positive sense. PEV are widely distributed in pig and wild boar. Infections are in most cases of subclinical nature (Fenner et al., 1993; Nardelli et al., 1993). Occasionally clinical signs develop which are attributed to PEV alone or may be caused by interaction with other pathogens. PEV infections have been reported to be associated with polioencephalomyelitis, reproductive disorders (SMEDI syndrome), pneumonia, and diarrhea (Dunne et al., 1965; Liebke and Schlenstedt, 1971; Edington et al., 1972). They usually do not cause heavy economic losses, even though nervous disorders called Teschen disease spread just like a harmful epidemic in most European countries in the 1940s and 1950s. The condition was initially observed in Czechoslovakia in 1929 (Nardelli et al., 1993). Subsequently, a less severe clinical syndrome occurred almost exclusively, firstly described in the United Kingdom and termed Talfan disease (Harding and Done, 1957). Teschen-Talfan disease was causually connected with infections by strains of PEV-1. More recently other serotypes have also been isolated from pigs with signs of polioencephalomyelitis which were associated with high morbidity and mortality (Witte et al., 1986; Appel et al., 1995). However, no serologic or molecular markers for differentiation of PEV strains corresponding to their virulence are available so far. Polioencephalomyelitis due to porcine enterovirus cannot be differentiated by pathologicanatomical signs alone from other viral encephalomyelitides, such as African swine fever, pseudorabies, hemagglutinating encephalomyelitis, rabies, and hog cholera. A final diagnosis requires the detection of virus (Fenner et al., 1993). Diagnosis of PEV is currently ascertained by isolation in cell culture followed by neutralization typing (NT) (Dunne et al., 1971; Knowles et al., 1979) or complement fixation test (Knowles and Buckley, 1980). These methods are time-consuming and labour-intensive. The interpretation of tests may be impaired if the virus replicates at low titres in cell culture or mixtures of viral types occur. Indirect immunofluorescence (IIF) and immunoperoxidase (IIP) staining can be used for grouping of PEV, provided production and selection of suitable sera are successful (Honda et al., 1990a; Auerbach et al., 1994). Because of pronounced cross-reactivity within serotype groups, typing with polyclonal sera by means of IIF and IIP is virtually impossible. Monoclonal antibodies (mAbs) may be a suitable tool to overcome these problems. In this paper, preliminary results are presented on the development of mAbs for antigenic characterization of PEV group I strains. 2. Materials and methods 2.1. Virus and cell cultures The enterovirus strains used in this study are reference strains and isolates collected from animals with clinical features as listed in Table 1. Swine vesicular disease virus
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Table 1 Virus strains and field isolates utilized for the development of anti-PEV monoclonal antibodies and the determination of antibody reactivity Virus *
PEV-1 PEV-1 PEV-1 PEV-1* PEV-1 PEV-1 PEV-1 PEV-? PEV-? PEV-2 PEV-2 PEV-2 PEV-2 PEV-2 PEV-2? PEV-3 PEV-4 PEV-4 PEV-4 PEV-4 PEV-5 PEV-5 PEV-6 PEV-6 PEV-6 PEV-7 PEV-7 PEV-11 PEV-11 PEV-12 PEV-13 PEV-8 PEV-9 PEV-10 SVDV BEV-1 FMDV-A5 FMDV-O1 FMDV-C1 EMCV
Strain/isolate
Reference
Source
Konratice PS 34 Teschen 199 Tirol Talfan 1626/89 DS 562/91 Dresden DS 1696/91 O3b T80 DS 334/93 DS 186/92 DS 804/92 DS 183/93 O2b PS 36 3764/86 918-19/85 E3/95/1 F26 1806/89 PS 37 3634/85 289/89 WR 2 F43 UKG 173/74 DS 805/92 2899/84 460/88 V 13 UKG 410/73 LP 54 UKG 27/72 LC R4 Riems Lausanne Teterow LC75
Edington et al. (1972) Dunne et al. (1965) Mayr unpubl. Mayr unpubl. Harding and Done (1957) Witte et al. (1994) Field isolate Hahnefeld et al. (1965) Appel et al. (1995) Kasza and Adler (1965) Betts (1960) Field isolate Field isolate Field isolate Field isolate Kasza and Adler (1965) Dunne et al. (1965) Witte et al. (1994) Witte et al. (1994) Field isolate Alexander and Betts (1967) Witte et al. (1994) Dunne et al. (1965) Witte et al. (1994) Witte et al. (1994) McConnel et al. (1968) Alexander and Betts (1967) Knowles et al. (1979) Field isolate Auerbach et al. (1994) Auerbach et al. (1994) Lamont and Betts (1960) Knowles et al. (1979) Knowles et al. (1979) Armstrong and Barnett (1989) Kunin and Minuse (1958) unpubl. Fontaine et al. (1968) unpubl. Nat. Inst. Vet. Med., Havana, Cuba
Hahnefeld, H.a ATCC Ahl, R.b Ahl, R. Ahl, R. Witte, K.H.c Steinhagen, P.d Hahnefeld, H. Steinhagen, P. ATCC Ahl, R. Steinhagen, P. Steinhagen, P. Steinhagen, P. Steinhagen, P. ATCC ATCC Witte, K.H. Witte, K.H. Kaden, V.e ATCC Witte, K.H. ATCC Witte, K.H. Witte, K.H. ATCC Ahl, R. Ahl, R. Steinhagen, P. Witte, K.H. Witte, K.H. Ahl, R. Ahl, R. Ahl, R. Kaden, V. Bergmann, H.a Kaden, V. Kaden, V. Kaden, V.
Note: The PEV strains employed for immunization are indicated by asterisk (*). Friedrich-Loeffler-Institut fuÈr Tierseuchenforschung Insel Riems. b Bundesforschungsanstalt fuÈr Viruskrankheiten der Tiere, TuÈbingen. c Staatliches VeterinaÈruntersuchungsamt, Arnsberg. d Lebensmittel- und VeterinaÈruntersuchungsamt, NeumuÈnster. e Bundesforschungsanstalt fuÈr Viruskrankheiten der Tiere, Insel Riems. a
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(SVDV), foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), and bovine enterovirus (BEV) were used as controls. PEVand EMCV were propagated in porcine embryonic testes cells (EFH), SVDV in embryonic pig kidney cells (EFN), BEV in bovine fetal lung cells (KLu), and FMDV in BHK-21/CT. (Cells were kindly supplied by R. Riebe, Collection of cell lines in veterinary medicine, Federal Research Centre for Virus Diseases of Animals, Insel Riems.) PEV-1 strains Konratice and Tirol were used for immunization of mice. The virus was harvested after complete destruction of the cell monolayer. After cycles of freezing and thawing and two homogenization steps with chloroform, the virus was concentrated by precipitation with 8% PEG 6000 for 16 h at 48C. Precipitates were sedimented by centrifugation, resuspended in Tris±EDTA±NaCl (TEN) buffer (0.02 M TrisHCl, 0.001 M EDTA, 0.15 M NaCl, pH 7.4±7.6), and finally pelleted by ultracentrifugation for 3 h at 68,000 g in a fixed-angle rotor. The infectivity of the concentrated virus was determined and its purity controlled by electron microscopy. Furthermore, the virus strain Tirol was purified by CsCl gradient (1.1±1.5 g/cm3) centrifugation for use in immuno electron microscopy, Western immunoblot, and immunoprecipitation. 2.2. Hybridoma cell preparation Female BALB/c mice were immunized with 150 ml of concentrated infectious virus (108 TCID50/50 ml) suspended in complete Freund's adjuvant (FA) by subcutanous and intraperitoneal (i.p.) route. The same amount of virus was given i.p. 50±70 days later mixed with incomplete FA. Within 3±6 months a third i.p. immunization with virus, but without adjuvant, was performed and 3 days later the mouse spleen cells were fused with SP2/0 myeloma cells. Various numbers of fused cells were seeded in order to select hybridomas which produce anti-PEV antibodies. Hybridoma supernatants were screened by IIF using PEV-1 infected cells. Screening on cells infected with all other serotypes was followed when the reaction with PEV-1 was positive. 2.3. Indirect immunofluorescence IIF was carried out on cells grown on 10 well multispot slides. The wells were seeded with EFH cells and the cells were allowed to form a monolayer which was then infected with virus at a dilution of 1 : 20 to 1 : 100 and incubated until slight CPE was observed, generally 16±20 h p.i. The cells were washed in isotonic buffer and air-dried before immersing the slides in acetone at room temperature (RT) for 15 min. 25 ml of the hybridoma culture supernatant was applied to a well and incubated at RT for 1 h. An indocarbocyanin conjugated goat anti-mouse IgG (H L) secondary antibody (Dianova GmbH) was used to detect the presence of specific murine anti PEV antibodies. For evaluation the fluorescence intensity was rated from not detectable (±) to strong () according to microscopical examination. 2.4. Neutralization test Supernatants of clones which secreted PEV specific antibodies were tested by titre reduction assay for their neutralizing capability in relation to irrelevant supernatants. 96-
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well culture plates were seeded with EFH cells and incubated at 378C in a 5% CO2 atmosphere until a confluent monolayer was formed. 10-fold dilutions of the virus were incubated with hybridoma supernatants for 1 h at 378C, then the culture fluid was replaced by these mixtures. Cells were observed for CPE daily up to 10 days p.i. and infectivity titres were determined according to Spearman and Kaerber (Mayr et al., 1974). The neutralization indices were calculated as negative decadal logarithms derived from the infectivity titres. A difference of log neutralization indices 1.7 was considered as significant (Mayr et al., 1977). 2.5. Western blot and immunoprecipitation assay Immunostaining of Western blots and immunoprecipitation assays were performed to identify the protein specificity of selected mAbs. Purified virions were dissociated with lysis buffer containing 5% SDS and the proteins were separated by SDS gel electrophoresis on 12% polyacrylamide gels (PAGE). Then the proteins were transferred to nitrocellulose (NC) membranes by electroelution and residual binding sites blocked with 5% bovine serum albumin in Tris buffered saline. Strips of the NC membranes were incubated with mAbs to be tested, an irrelevant mAb and a virus specific polyclonal antiserum. After washing the specimens were incubated with goat anti-mouse IgG (H L) horseradish peroxidase conjugate (Bio-Rad Laboratories). The immunoblots were developed by chemoluminescence (ECL kit, Amersham International). To prepare all lysates for immunoprecipitation monolayers of EFH cells were infected with PEV and scraped off when about 75% of cells showed CPE. The cells were centrifuged and washed twice in PBS, resuspended in lysis buffer (sodium borate 50 mM, NaCl 150 mM, PMSF 0.1 mg/ml, aprotinin 1 mg/ml, leupeptin 1 mg/ml, Nonidet P-40 1%, sodium deoxycholate 0.5%). The lysate was frozen and homogenized after thawing. In some experiments, SDS up to 1% was added to the lysis buffer and removed again by dialysis from the cell lysate after the homogenization step. Then the preparation was biotin-labeled, cell fragments precipitated with protein A-agarose and the supernatant containing virus proteins incubated with the antibodies in question. Immunoprecipitates were obtained by incubation with protein A-agarose according to instructions of the cellular labeling and immunoprecipitation kit (Boehringer Mannheim), separated in PAGE, blotted on NC membranes, and marked with streptavidin-peroxidase conjugate (Boehringer Mannheim). Visualization was done by chemoluminescence. For verification of results, virus strain Tirol purified by CsCl gradient centrifugation was used. 3. Results and discussion As a final result eight hybridomas were obtained, which produced continuously IgG antibodies against PEV-1. These hybridomas were selected out of 516 hybridoma lines, which were cultivated from aliquots of fused cells derived from spleens of three immunized mice. Two fusions were performed using mice immunized with strain Konratice and one fusion with strain Tirol. Six of these eight mAbs reacted in IIF not only with cells infected by homologous or other PEV-1 strains, but showed a more or less
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Table 2 Reactivity of monoclonal antibodies induced against PEV-1 strains in indirect immunofluorescence PEV
Strain
1 1 1 1 1 1 1 ? ? 2 2 2 2 2 2? 3 4 4 4 4 5 5 6 6 6 7 7 11 11 12 13 8 9 10
Konraticea PS 34 Teschen 199 Tirolb Talfan 1626/89 DS 562/91 Dresden DS 1696/91 O3b T80 DS 334/93 DS 186/92 DS 804/92 DS 183/93 O2b PS 36 3764/86 918-19/85 E3/95/1 F26 1806/89 PS 37 3634/85 289/89 WR-2 F43 UKG 173/74 805/92 2899/84 460/88 V-13 UKG 410/73 LP54
Monoclonal antibody 040/4B1 040/5E9
040/7C9
158/10B8 158/11B5 158/3G4 158/5D2 158/5F1
± ± ±
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Fluorescence intensity: not detectable (±); weak (); medium (); strong (). Strain used for derivation of mAbs 040/-. For mAbs 158/-.
a
b
pronounced cross-reactivity to further serotypes with the exception of PEV-8, 9 and 10 strains. An overview of reactivity patterns is shown in Table 2. Only two mAbs, 158/5D2 and 158/5F1 recognized PEV-1 strains alone without such a cross-reaction with other serotypes. Both mAbs are homologous to strain Tirol. Remarkably, 158/5F1 did not react with all heterologous PEV-1 strains in comparable strength. In particular, the fluorescence with strain Konratice was very weak. Group-reactive epitopes of enteroviruses appear to be of poor immunogenicity. Only one mAb, 040/4B1, homologous to strain Konratice, reacted with all PEV serotypes of
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group I (PEV1-7,11-13) in strong intensity. This conclusion is supported by the observation, that none of 30 other mAbs, which were homologous to different PEV serotypes of group I, reacted with all virus strains of PEV group I despite strong crossreactivity (data not shown). On the other hand, strong serotype specificity appears to be rather an exception too. These observations agree with previous data on a group-specific mAb against human enteroviruses (Yousef et al., 1987). Using the group-specific mAb 040/4B1, the identity of PEV has been proved beyond doubt by IIF screening of reference strains comprising all serotypes which belong to group I. Many field isolates of different serotypes originating from pigs showing signs of polioencephalomyelitis or with the suspicion of hog cholera could be identified as PEV (Witte et al., 1994; Appel et al., 1995; Kaden, unpubl.; Steinhagen, unpubl.; see Table 1 and Table 2). Mixed infection with different PEV serotypes such as DS 183/93 isolate (Table 1) was no problem for diagnosis. In order to demonstrate the specificity of mAb 040/4B1 for PEV the possibility of cross-reactions was tested with the following picornaviruses: swine vesicular disease virus, bovine enterovirus 1, foot-and-mouth disease virus O1, A5 and C1, encephalomyocarditis virus. None of these viruses showed any reaction with this mAb in IIF or, in case of SVDV and FMDV, in IIP. With regard to mAb 158/5D2, further investigations are necessary to clarify if the mAb can be used to identify PEV serotype 1 field isolates unequivocally. For this purpose, other mAbs displaying serotype specificity should be included and the selection of further mAbs is envisaged. Immunofluorescence of PEV group I infected cells based on these mAbs is characterized by a pronounced cytoplasmic reaction, the staining pattern appearing often coarse-grained and of increased intensity near the nucleus (Fig. 1). The infected cells round up quickly as an effect of virus replication with the consequence that their fluorescence staining pattern is difficult to differentiate from that of noninfected rounded cells. However, mAb 040/4B1 yields with PEV infected cells a pronounced reaction, thus a differentiation from weak or unspecific fluorescence is no problem. Results of IgG isotyping and of virus neutralization by mAbs are summarized in Table 3. There were only two mAbs, 158/3G4 and 158/5D2, which neutralized the tested Table 3 Origin and properties of monoclonal antibodies against PEV: IgG subtypes, virus neutralization, and reactivity in immuno electron microscopy (IEM) mAb
Homologous virus strain
IgG
Virus neutralization
IEM
040/4B1 040/5E9 040/7C9 158/10B8 158/11B5 158/3G4 158/5D2 158/5F1
Konratice Konratice Konratice Tirol Tirol Tirol Tirol Tirol
2a 1 1 1 1 2a 2a 2a
± ± ± ± ± ±
± nt nt nt nt ±
nt: Not tested.
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M. Dauber / Veterinary Microbiology 67 (1999) 1±12
Fig. 1. PEV-1 infected porcine embryonic tested cells (EFH) examined by IIF with mAb 040/4B1. Goat antimouse IgG (H L) indocarbocyanin, bar 50 mm.
PEV-1 strains Konratice, Teschen 199 and Tirol significantly. It is remarkable that especially these two mAbs reacted with virions in immuno electron microscopy out of four mAbs tested (Granzow, pers. comm., see Fig. 2). The two nonreacting mAbs were also not able to neutralize virus. In Western immunoblot assays mAbs 040/4B1 and 158/5D2 did not react at all (data not shown). However, by immunoprecipitation these mAbs reacted with three proteins of about 37, 30 and 28 kDa. The proteins probably represent the viral capsid proteins VP1, VP2 and VP3. It was not possible to detect a reaction with a single protein alone. We assume, that the lysis of virus-containing cells prior to precipitation did not result in a complete dissociation of virus particles. Consequently, mAbs precipitated either whole virions or empty capsids which were only disintegrated in the next step by suspension in
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Fig. 2. Reaction of porcine enterovirus 1 strain Tirol with monoclonal antibodies 040/4B1 (a) and 158/5D2 (b). No specific labeling of virus particles by mAb 040/4B1 in contrast to specific labeling with 158/5D2. Goat-anti mouse IgG immunogold 10 nm (BioCell Int., Cambridge), bar 150 nm. Immuno electron microscopy by courtesy of Dr. H. Granzow.
PAGE lysis buffer. The addition of up to 0.1% SDS to the immunoprecipitation lysis buffer had no effect. A further increase of the SDS content abolished precipitation completely. In another approach lysis of virions with buffer containing 1% SDS was then followed by dialysis in order to remove SDS prior to precipitation. However, the reaction pattern was identical to that obtained after mild lysis conditions (Fig. 3). These results correlate with the findings of Wiley et al. (1990, 1992), concerning the reactivity of mAbs against human enterovirus EV-70 in immunoblot and radioimmunoprecipitation (RIP) assays. Obviously, these mAbs bind to conformational viral epitopes because they do not react in immunoblot and by immunoprecipitation three proteins were always displayed together. The question remains open whether the mAbs react with epitopes located on one protein only or epitopes shaped by association of two or three proteins. Both the mAbs 158/5D2 and 040/4B1 reacted similarly in immunoblot and immunoprecipitation. However, in contrast to 040/4B1 the mAb 158/5D2 is able to
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Fig. 3. Chemoluminescence patterns of PEV-1 (strain Tirol) proteins precipitated by group-specific mAb 040/ 4B1 and serotype specific mAb 158/5D2. Virus was purified by ultracentrifugation on CsCl gradients and suspended in lysis buffer without SDS (A) and in lysis buffer with 1% SDS that was then removed by dialysis, respectively (B). The proteins were labeled with biotin and then precipitated by antibodies. Lane 1: rabbit antiPEV-1 serum, Lane 2: mAb 040/4B1, Lane 3: mAb 158/5D2, Lane 4: irrelevant mAb 041/3C3 (specific to another PEV serotype). Precipitates were separated by SDS-PAGE and blotted on NC membranes. M: calibration proteins. Arrows indicate specific reactions at about 37, 30 and 28 kDa, probably representing virus proteins VP1, VP2 and VP3. There is no reaction with a single protein alone.
neutralize virus. These data indicate that both mAbs recognize three-dimensional structures. It appears that mAb 040/4B1 binds to a conserved region located within the virion because of its reactivity with many serotypes and lack of neutralizing activity. Contrarily, the neutralizing mAb 158/5D2 reacts in IEM, therefore, the corresponding epitopes must be located on the outer surface. In order to diagnose PEV, the application of the group-specific mAb 040/4B1 instead of antisera will be an advantage, because antibodies to irrelevant antigens are not present. It is not possible to standardize polyclonal antisera. This procedure requires many tests with reference probes, high background staining can occur and only low dilutions are applicable. These problems may lead to wrong interpretations of results. In comparison with sera, application of these mAbs is useful for a rapid detection of group I PEV by IIF in a highly reproducible manner. Acknowledgements I am grateful to R. Ahl, P. Steinhagen and K.H. Witte who kindly supplied porcine enterovirus strains and isolates. V. Kaden carried out tests with SVDV and FMDV and H. Granzow provided the electronmicrographs for this publication. I thank Eveline Ball and
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Hanna Wege for excellent technical assistance and Helmut Wege for stimulating discussion. References Alexander, T.J.L., Betts, A.O., 1967. Further studies on porcine enteroviruses isolated at Cambridge I. Serological grouping. Res. Vet. Sci. 8, 330±337. Appel, G., Steinhagen, P., Ohlinger, V.F., Ewald, C., 1995. Enzephalomyelopathie bei sauen und mastschweinen infolge einer infektion mit porzinem enterovirus (PEV). TieraÈrztl. Umschau 50, 326±336. Armstrong, R.M., Barnett, I.T.R., 1989. An enzyme linked immunosorbent assay (ELISA) for the detection and quantification of antibodies against swine vesicular disease virus (SVDV). J. Virol. Methods 25, 71± 80. Auerbach, J., Prager, D., Neuhaus, S., Loss, U., Witte, K.H., 1994. Grouping of porcine enteroviruses by indirect immunofluorescence and description of two new serotypes. J. Vet. Med. B 41, 277±282. Betts, A.O., 1960. Studies on enteroviruses of the pig ± the recovery in tissue culture of two related strains of swine polioencephalomyelitis virus from the tonsils of `normal' pigs. Res. Vet. Sci. 1, 57±64. Dunne, H.W., Gobble, J.L., Hokanson, J.F., Kradel, D.C., Bubash, G.R., 1965. Porcine reproductive failure associated with a newly identified `SMEDI' group of picorna viruses. Am. J. Vet Res. 26, 1284±1297. Dunne, H.W., Wang, J.T., Ammerman, E.H., 1971. Classification of North American porcine enteroviruses: a comparison with European and Japanese strains. Infec. Immun. 4, 619±631. Edington, N., Christofinis, G.J., Betts, A.O., 1972. Pathogenicity of Talfan and Konratice strains of Teschen virus in gnotobiotic pigs. J. Comp. Path. 82, 393±399. Fenner, F.J., Gibbs, E.P.J., Murphy, F.A., Rott, R., Studdert, M.J., White, D.O., 1993. Porcine enteroviruses. In: Veterinary Virology, Academic Press, San Diego, pp. 416±419. Fontaine, J., Mackowiak, C., Roumiantzeff, M., 1968. Types, sous-types et variantes du virus aphteux etude des variants. Symp. Series Immunobiol. Standard 8, 13±64. Hahnefeld, H., Hahnefeld, E., Wittig, W., 1965. Talfan disease in Deutschland 1. Mitteilung: Isolierung und Charakterisierung von Teschenvirus Subtyp Talfan bei Saugferkeln im Bezirk Dresden. Arch. exp. VeterinaÈrmed. 19, 185±218. Harding, J.D.J., Done, J.T., 1957. A transmissible polioencephalomyelitis of pigs (Talfan disease). Vet. Rec. 69, 2±8. Honda, E., Kimata, A., Hattori, I., Kumagai, T., Tsuda, T., Tokui, T., 1990. A serological comparison of four Japanese isolates of porcine enteroviruses with the international reference strains. Jpn. J. Vet. Sci. 52, 49± 54. Honda, E., Watanabe, I., Okazaki, K., Kumagai, T., 1990a. Relation of serological and CPE-classification of porcine enteroviruses to the classification by immunoperoxidase (IP) staining, and observation of CPE by IP staining method. Jpn. J. Vet. Sci. 52, 795±800. Kasza, L., Adler, A., 1965. Biologic and immunologic characterization of six swine enterovirus isolates. Am. J. Vet. Sci. 26, 625±630. Knowles, N.J., Buckley, L.S., 1980. Differentiation of porcine enterovirus serotypes by complement fixation. Res. Vet. Sci. 29, 113±115. Knowles, N.J., 1988. The association of group III porcine enteroviruses with epithelial tissue. Vet. Rec. 122, 441±442. Knowles, N.J., Buckley, L.S., Pereira, H.G., 1979. Classification of porcine enteroviruses by antigenic analysis and cythopathic effects in tissue culture: description of three new serotypes. Arch. Virol. 62, 201±208. Kunin, C.M., Minuse, E., 1958. The isolation in tissue culture, chick embryo and suckling mice of filtrable agents from healthy dairy cattle. J. Immunol. 80, 1±11. Lamont, P.H., Betts, A.O., 1960. Studies on enteroviruses of the pig IV. The isolation in tissue culture of a possible enteric cytopathogenic swine orphan (ECSO) virus (V 13) from the feces of a pig. Res. Vet. Sci. 1, 152±159. Liebke, H., Schlenstedt, D., 1971. Eine Enterovirus (ECSO)-Infektion bei Schweinen mit nervoÈsen StoÈrungen und einer gleichzeitig vorhandenen Rhinitis. TieraÈrztl. Umschau, 26, 287±291; 324±330.
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Mayr, A., Bachmann, P.A., Bibrack, B., Wittmann, G., 1974. Virologische Arbeitsmethoden. Bd. I, VEB Gustav Fischer Verlag, Jena, p. 39. Mayr, A., Bachmann, P.A., Bibrack, B., Wittmann, G., 1977. Virologische Arbeitsmethoden. Bd. II, VEB Gustav Fischer Verlag, Jena, pp. 469±473. McConnel, S., Spertzel, R.O., Shively, J.N., 1968. Isolation, characterization, and serologic comparison of selected porcine enteroviruses by plaque reduction test. Am. J. Vet. Res. 29, 245±251. Minor, P.D., Brown, F., Domingo, E., Hoey, E., King, A., Knowles, N., Lemon, S., Palmenberg, A., Rueckert, R.R., Stanway, G., Wimmer, E., Yin-Murphy, M., 1995. Family Picornaviridae. In: Murphy, F.A., Fauquet, C.M., Bishop, D.H.L., Ghabrial, S.H., Jarvis, A.W., Martelli, G.P., Mayo, M.A., Summers M.D. (Eds.), Virus Taxonomy. Classification and Nomenclature of Viruses. 6th Report of the Int. Committee on Taxonomy of Viruses. Arch. Virol. Suppl. 10, 329±336. Nardelli, S., Farina, L., Selli, L., Parpaiola, R., Zuin, A., 1993. Research on the presence of porcine enterovirus serotype 1 in northeastern Italy. J. Vet. Med. B 40, 190±196. Witte, K.H., Auerbach, J., Loss, K.U., Neuhaus, S., Prager, D., 1994. Typisierung von 17 porzinen Enterovirusisolaten aus PolioenzephalomyelitisfaÈllen der Jahre 1983±1991. Dtsch. TieraÈrztl. Wschr. 101, 453±492. Witte, K.H., Prager, D., Schoon, H.-A., Woicke, J., Hertrampf, B., 1986. Durch porzines Enterovirus Serotyp 2 verursachte verlustreiche Polioenzephalomyelitis in einem Schweinemastbestand. Berl. MuÈnch. TieraÈrztl. Wschr. 99, 121±127. Wiley, J.A., Brodeur, B.R., Dimock, K.D., Sattar, S.A., 1990. Neutralizing monoclonal antibody against enterovirus-70 reacts with viral proteins IC and ID. Viral Immunol. 3, 137±146. Wiley, J.A., Hamel, J., Brodeur, B.R., 1992. Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes. J. Virol. 66, 5744±5751. Yousef, G.E., Mann, G.F., Brown, I.N., Mowbray, J.F., 1987. Clinical and research appplication of an enterovirus group-reactive monoclonal antibody. Intervirology 28, 199±205.
Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture M. Dauber* Federal Research Centre for Virus Diseases of Animals, Friedrich Loeffler Institutes, Institute of Diagnostic Virology, Boddenblick 5a, D-17498 Insel Riems, Germany Received 18 September 1998; accepted 15 February 1999
Abstract A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Pig-viruses; Porcine enterovirus; Monoclonal antibody; Diagnosis; Immuno¯uorescence
1. Introduction Porcine enteroviruses (PEV) have been classified to the genus Enterovirus of the family Picornaviridae (Honda et al., 1990; Auerbach et al., 1994; Minor et al., 1995) and at least 13 species (serotypes) can be distinguished. Classification of PEV is primarily based on a minimum relationship indicated by serum neutralization titre determined in cross-reaction tests. When the heterologous titre of a PEV reaches at least 5% of the homologous one both strains are assigned to the same serotype (Dunne et al., 1971; Knowles et al., 1979). Three groups of PEV can be distinguished by parameters such as physico-chemical properties, type of cytopathic effect in pig kidney cells, cross-reactions, and genomic conformity. Most of these viruses are integrated into serotype group I * Tel.: +49-38351-70; fax: +49-38351-7219; e-mail: [email protected] 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 2 4 - 3
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(PEV1-7, 11-13), which have many characteristic features in common but differ in some points from all the other enteroviruses (Knowles, 1988). PEV-8 represents group II and serotypes 9 and 10 form group III. The virions are icosahedral particles and have a 28± 30 nm capsid without envelope, made up of 60 copies of four proteins, VP1 to VP4. The genome consists of a single stranded monopartite RNA with positive sense. PEV are widely distributed in pig and wild boar. Infections are in most cases of subclinical nature (Fenner et al., 1993; Nardelli et al., 1993). Occasionally clinical signs develop which are attributed to PEV alone or may be caused by interaction with other pathogens. PEV infections have been reported to be associated with polioencephalomyelitis, reproductive disorders (SMEDI syndrome), pneumonia, and diarrhea (Dunne et al., 1965; Liebke and Schlenstedt, 1971; Edington et al., 1972). They usually do not cause heavy economic losses, even though nervous disorders called Teschen disease spread just like a harmful epidemic in most European countries in the 1940s and 1950s. The condition was initially observed in Czechoslovakia in 1929 (Nardelli et al., 1993). Subsequently, a less severe clinical syndrome occurred almost exclusively, firstly described in the United Kingdom and termed Talfan disease (Harding and Done, 1957). Teschen-Talfan disease was causually connected with infections by strains of PEV-1. More recently other serotypes have also been isolated from pigs with signs of polioencephalomyelitis which were associated with high morbidity and mortality (Witte et al., 1986; Appel et al., 1995). However, no serologic or molecular markers for differentiation of PEV strains corresponding to their virulence are available so far. Polioencephalomyelitis due to porcine enterovirus cannot be differentiated by pathologicanatomical signs alone from other viral encephalomyelitides, such as African swine fever, pseudorabies, hemagglutinating encephalomyelitis, rabies, and hog cholera. A final diagnosis requires the detection of virus (Fenner et al., 1993). Diagnosis of PEV is currently ascertained by isolation in cell culture followed by neutralization typing (NT) (Dunne et al., 1971; Knowles et al., 1979) or complement fixation test (Knowles and Buckley, 1980). These methods are time-consuming and labour-intensive. The interpretation of tests may be impaired if the virus replicates at low titres in cell culture or mixtures of viral types occur. Indirect immunofluorescence (IIF) and immunoperoxidase (IIP) staining can be used for grouping of PEV, provided production and selection of suitable sera are successful (Honda et al., 1990a; Auerbach et al., 1994). Because of pronounced cross-reactivity within serotype groups, typing with polyclonal sera by means of IIF and IIP is virtually impossible. Monoclonal antibodies (mAbs) may be a suitable tool to overcome these problems. In this paper, preliminary results are presented on the development of mAbs for antigenic characterization of PEV group I strains. 2. Materials and methods 2.1. Virus and cell cultures The enterovirus strains used in this study are reference strains and isolates collected from animals with clinical features as listed in Table 1. Swine vesicular disease virus
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Table 1 Virus strains and field isolates utilized for the development of anti-PEV monoclonal antibodies and the determination of antibody reactivity Virus *
PEV-1 PEV-1 PEV-1 PEV-1* PEV-1 PEV-1 PEV-1 PEV-? PEV-? PEV-2 PEV-2 PEV-2 PEV-2 PEV-2 PEV-2? PEV-3 PEV-4 PEV-4 PEV-4 PEV-4 PEV-5 PEV-5 PEV-6 PEV-6 PEV-6 PEV-7 PEV-7 PEV-11 PEV-11 PEV-12 PEV-13 PEV-8 PEV-9 PEV-10 SVDV BEV-1 FMDV-A5 FMDV-O1 FMDV-C1 EMCV
Strain/isolate
Reference
Source
Konratice PS 34 Teschen 199 Tirol Talfan 1626/89 DS 562/91 Dresden DS 1696/91 O3b T80 DS 334/93 DS 186/92 DS 804/92 DS 183/93 O2b PS 36 3764/86 918-19/85 E3/95/1 F26 1806/89 PS 37 3634/85 289/89 WR 2 F43 UKG 173/74 DS 805/92 2899/84 460/88 V 13 UKG 410/73 LP 54 UKG 27/72 LC R4 Riems Lausanne Teterow LC75
Edington et al. (1972) Dunne et al. (1965) Mayr unpubl. Mayr unpubl. Harding and Done (1957) Witte et al. (1994) Field isolate Hahnefeld et al. (1965) Appel et al. (1995) Kasza and Adler (1965) Betts (1960) Field isolate Field isolate Field isolate Field isolate Kasza and Adler (1965) Dunne et al. (1965) Witte et al. (1994) Witte et al. (1994) Field isolate Alexander and Betts (1967) Witte et al. (1994) Dunne et al. (1965) Witte et al. (1994) Witte et al. (1994) McConnel et al. (1968) Alexander and Betts (1967) Knowles et al. (1979) Field isolate Auerbach et al. (1994) Auerbach et al. (1994) Lamont and Betts (1960) Knowles et al. (1979) Knowles et al. (1979) Armstrong and Barnett (1989) Kunin and Minuse (1958) unpubl. Fontaine et al. (1968) unpubl. Nat. Inst. Vet. Med., Havana, Cuba
Hahnefeld, H.a ATCC Ahl, R.b Ahl, R. Ahl, R. Witte, K.H.c Steinhagen, P.d Hahnefeld, H. Steinhagen, P. ATCC Ahl, R. Steinhagen, P. Steinhagen, P. Steinhagen, P. Steinhagen, P. ATCC ATCC Witte, K.H. Witte, K.H. Kaden, V.e ATCC Witte, K.H. ATCC Witte, K.H. Witte, K.H. ATCC Ahl, R. Ahl, R. Steinhagen, P. Witte, K.H. Witte, K.H. Ahl, R. Ahl, R. Ahl, R. Kaden, V. Bergmann, H.a Kaden, V. Kaden, V. Kaden, V.
Note: The PEV strains employed for immunization are indicated by asterisk (*). Friedrich-Loeffler-Institut fuÈr Tierseuchenforschung Insel Riems. b Bundesforschungsanstalt fuÈr Viruskrankheiten der Tiere, TuÈbingen. c Staatliches VeterinaÈruntersuchungsamt, Arnsberg. d Lebensmittel- und VeterinaÈruntersuchungsamt, NeumuÈnster. e Bundesforschungsanstalt fuÈr Viruskrankheiten der Tiere, Insel Riems. a
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(SVDV), foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), and bovine enterovirus (BEV) were used as controls. PEVand EMCV were propagated in porcine embryonic testes cells (EFH), SVDV in embryonic pig kidney cells (EFN), BEV in bovine fetal lung cells (KLu), and FMDV in BHK-21/CT. (Cells were kindly supplied by R. Riebe, Collection of cell lines in veterinary medicine, Federal Research Centre for Virus Diseases of Animals, Insel Riems.) PEV-1 strains Konratice and Tirol were used for immunization of mice. The virus was harvested after complete destruction of the cell monolayer. After cycles of freezing and thawing and two homogenization steps with chloroform, the virus was concentrated by precipitation with 8% PEG 6000 for 16 h at 48C. Precipitates were sedimented by centrifugation, resuspended in Tris±EDTA±NaCl (TEN) buffer (0.02 M TrisHCl, 0.001 M EDTA, 0.15 M NaCl, pH 7.4±7.6), and finally pelleted by ultracentrifugation for 3 h at 68,000 g in a fixed-angle rotor. The infectivity of the concentrated virus was determined and its purity controlled by electron microscopy. Furthermore, the virus strain Tirol was purified by CsCl gradient (1.1±1.5 g/cm3) centrifugation for use in immuno electron microscopy, Western immunoblot, and immunoprecipitation. 2.2. Hybridoma cell preparation Female BALB/c mice were immunized with 150 ml of concentrated infectious virus (108 TCID50/50 ml) suspended in complete Freund's adjuvant (FA) by subcutanous and intraperitoneal (i.p.) route. The same amount of virus was given i.p. 50±70 days later mixed with incomplete FA. Within 3±6 months a third i.p. immunization with virus, but without adjuvant, was performed and 3 days later the mouse spleen cells were fused with SP2/0 myeloma cells. Various numbers of fused cells were seeded in order to select hybridomas which produce anti-PEV antibodies. Hybridoma supernatants were screened by IIF using PEV-1 infected cells. Screening on cells infected with all other serotypes was followed when the reaction with PEV-1 was positive. 2.3. Indirect immunofluorescence IIF was carried out on cells grown on 10 well multispot slides. The wells were seeded with EFH cells and the cells were allowed to form a monolayer which was then infected with virus at a dilution of 1 : 20 to 1 : 100 and incubated until slight CPE was observed, generally 16±20 h p.i. The cells were washed in isotonic buffer and air-dried before immersing the slides in acetone at room temperature (RT) for 15 min. 25 ml of the hybridoma culture supernatant was applied to a well and incubated at RT for 1 h. An indocarbocyanin conjugated goat anti-mouse IgG (H L) secondary antibody (Dianova GmbH) was used to detect the presence of specific murine anti PEV antibodies. For evaluation the fluorescence intensity was rated from not detectable (±) to strong () according to microscopical examination. 2.4. Neutralization test Supernatants of clones which secreted PEV specific antibodies were tested by titre reduction assay for their neutralizing capability in relation to irrelevant supernatants. 96-
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well culture plates were seeded with EFH cells and incubated at 378C in a 5% CO2 atmosphere until a confluent monolayer was formed. 10-fold dilutions of the virus were incubated with hybridoma supernatants for 1 h at 378C, then the culture fluid was replaced by these mixtures. Cells were observed for CPE daily up to 10 days p.i. and infectivity titres were determined according to Spearman and Kaerber (Mayr et al., 1974). The neutralization indices were calculated as negative decadal logarithms derived from the infectivity titres. A difference of log neutralization indices 1.7 was considered as significant (Mayr et al., 1977). 2.5. Western blot and immunoprecipitation assay Immunostaining of Western blots and immunoprecipitation assays were performed to identify the protein specificity of selected mAbs. Purified virions were dissociated with lysis buffer containing 5% SDS and the proteins were separated by SDS gel electrophoresis on 12% polyacrylamide gels (PAGE). Then the proteins were transferred to nitrocellulose (NC) membranes by electroelution and residual binding sites blocked with 5% bovine serum albumin in Tris buffered saline. Strips of the NC membranes were incubated with mAbs to be tested, an irrelevant mAb and a virus specific polyclonal antiserum. After washing the specimens were incubated with goat anti-mouse IgG (H L) horseradish peroxidase conjugate (Bio-Rad Laboratories). The immunoblots were developed by chemoluminescence (ECL kit, Amersham International). To prepare all lysates for immunoprecipitation monolayers of EFH cells were infected with PEV and scraped off when about 75% of cells showed CPE. The cells were centrifuged and washed twice in PBS, resuspended in lysis buffer (sodium borate 50 mM, NaCl 150 mM, PMSF 0.1 mg/ml, aprotinin 1 mg/ml, leupeptin 1 mg/ml, Nonidet P-40 1%, sodium deoxycholate 0.5%). The lysate was frozen and homogenized after thawing. In some experiments, SDS up to 1% was added to the lysis buffer and removed again by dialysis from the cell lysate after the homogenization step. Then the preparation was biotin-labeled, cell fragments precipitated with protein A-agarose and the supernatant containing virus proteins incubated with the antibodies in question. Immunoprecipitates were obtained by incubation with protein A-agarose according to instructions of the cellular labeling and immunoprecipitation kit (Boehringer Mannheim), separated in PAGE, blotted on NC membranes, and marked with streptavidin-peroxidase conjugate (Boehringer Mannheim). Visualization was done by chemoluminescence. For verification of results, virus strain Tirol purified by CsCl gradient centrifugation was used. 3. Results and discussion As a final result eight hybridomas were obtained, which produced continuously IgG antibodies against PEV-1. These hybridomas were selected out of 516 hybridoma lines, which were cultivated from aliquots of fused cells derived from spleens of three immunized mice. Two fusions were performed using mice immunized with strain Konratice and one fusion with strain Tirol. Six of these eight mAbs reacted in IIF not only with cells infected by homologous or other PEV-1 strains, but showed a more or less
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Table 2 Reactivity of monoclonal antibodies induced against PEV-1 strains in indirect immunofluorescence PEV
Strain
1 1 1 1 1 1 1 ? ? 2 2 2 2 2 2? 3 4 4 4 4 5 5 6 6 6 7 7 11 11 12 13 8 9 10
Konraticea PS 34 Teschen 199 Tirolb Talfan 1626/89 DS 562/91 Dresden DS 1696/91 O3b T80 DS 334/93 DS 186/92 DS 804/92 DS 183/93 O2b PS 36 3764/86 918-19/85 E3/95/1 F26 1806/89 PS 37 3634/85 289/89 WR-2 F43 UKG 173/74 805/92 2899/84 460/88 V-13 UKG 410/73 LP54
Monoclonal antibody 040/4B1 040/5E9
040/7C9
158/10B8 158/11B5 158/3G4 158/5D2 158/5F1
± ± ±
± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Fluorescence intensity: not detectable (±); weak (); medium (); strong (). Strain used for derivation of mAbs 040/-. For mAbs 158/-.
a
b
pronounced cross-reactivity to further serotypes with the exception of PEV-8, 9 and 10 strains. An overview of reactivity patterns is shown in Table 2. Only two mAbs, 158/5D2 and 158/5F1 recognized PEV-1 strains alone without such a cross-reaction with other serotypes. Both mAbs are homologous to strain Tirol. Remarkably, 158/5F1 did not react with all heterologous PEV-1 strains in comparable strength. In particular, the fluorescence with strain Konratice was very weak. Group-reactive epitopes of enteroviruses appear to be of poor immunogenicity. Only one mAb, 040/4B1, homologous to strain Konratice, reacted with all PEV serotypes of
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group I (PEV1-7,11-13) in strong intensity. This conclusion is supported by the observation, that none of 30 other mAbs, which were homologous to different PEV serotypes of group I, reacted with all virus strains of PEV group I despite strong crossreactivity (data not shown). On the other hand, strong serotype specificity appears to be rather an exception too. These observations agree with previous data on a group-specific mAb against human enteroviruses (Yousef et al., 1987). Using the group-specific mAb 040/4B1, the identity of PEV has been proved beyond doubt by IIF screening of reference strains comprising all serotypes which belong to group I. Many field isolates of different serotypes originating from pigs showing signs of polioencephalomyelitis or with the suspicion of hog cholera could be identified as PEV (Witte et al., 1994; Appel et al., 1995; Kaden, unpubl.; Steinhagen, unpubl.; see Table 1 and Table 2). Mixed infection with different PEV serotypes such as DS 183/93 isolate (Table 1) was no problem for diagnosis. In order to demonstrate the specificity of mAb 040/4B1 for PEV the possibility of cross-reactions was tested with the following picornaviruses: swine vesicular disease virus, bovine enterovirus 1, foot-and-mouth disease virus O1, A5 and C1, encephalomyocarditis virus. None of these viruses showed any reaction with this mAb in IIF or, in case of SVDV and FMDV, in IIP. With regard to mAb 158/5D2, further investigations are necessary to clarify if the mAb can be used to identify PEV serotype 1 field isolates unequivocally. For this purpose, other mAbs displaying serotype specificity should be included and the selection of further mAbs is envisaged. Immunofluorescence of PEV group I infected cells based on these mAbs is characterized by a pronounced cytoplasmic reaction, the staining pattern appearing often coarse-grained and of increased intensity near the nucleus (Fig. 1). The infected cells round up quickly as an effect of virus replication with the consequence that their fluorescence staining pattern is difficult to differentiate from that of noninfected rounded cells. However, mAb 040/4B1 yields with PEV infected cells a pronounced reaction, thus a differentiation from weak or unspecific fluorescence is no problem. Results of IgG isotyping and of virus neutralization by mAbs are summarized in Table 3. There were only two mAbs, 158/3G4 and 158/5D2, which neutralized the tested Table 3 Origin and properties of monoclonal antibodies against PEV: IgG subtypes, virus neutralization, and reactivity in immuno electron microscopy (IEM) mAb
Homologous virus strain
IgG
Virus neutralization
IEM
040/4B1 040/5E9 040/7C9 158/10B8 158/11B5 158/3G4 158/5D2 158/5F1
Konratice Konratice Konratice Tirol Tirol Tirol Tirol Tirol
2a 1 1 1 1 2a 2a 2a
± ± ± ± ± ±
± nt nt nt nt ±
nt: Not tested.
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Fig. 1. PEV-1 infected porcine embryonic tested cells (EFH) examined by IIF with mAb 040/4B1. Goat antimouse IgG (H L) indocarbocyanin, bar 50 mm.
PEV-1 strains Konratice, Teschen 199 and Tirol significantly. It is remarkable that especially these two mAbs reacted with virions in immuno electron microscopy out of four mAbs tested (Granzow, pers. comm., see Fig. 2). The two nonreacting mAbs were also not able to neutralize virus. In Western immunoblot assays mAbs 040/4B1 and 158/5D2 did not react at all (data not shown). However, by immunoprecipitation these mAbs reacted with three proteins of about 37, 30 and 28 kDa. The proteins probably represent the viral capsid proteins VP1, VP2 and VP3. It was not possible to detect a reaction with a single protein alone. We assume, that the lysis of virus-containing cells prior to precipitation did not result in a complete dissociation of virus particles. Consequently, mAbs precipitated either whole virions or empty capsids which were only disintegrated in the next step by suspension in
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Fig. 2. Reaction of porcine enterovirus 1 strain Tirol with monoclonal antibodies 040/4B1 (a) and 158/5D2 (b). No specific labeling of virus particles by mAb 040/4B1 in contrast to specific labeling with 158/5D2. Goat-anti mouse IgG immunogold 10 nm (BioCell Int., Cambridge), bar 150 nm. Immuno electron microscopy by courtesy of Dr. H. Granzow.
PAGE lysis buffer. The addition of up to 0.1% SDS to the immunoprecipitation lysis buffer had no effect. A further increase of the SDS content abolished precipitation completely. In another approach lysis of virions with buffer containing 1% SDS was then followed by dialysis in order to remove SDS prior to precipitation. However, the reaction pattern was identical to that obtained after mild lysis conditions (Fig. 3). These results correlate with the findings of Wiley et al. (1990, 1992), concerning the reactivity of mAbs against human enterovirus EV-70 in immunoblot and radioimmunoprecipitation (RIP) assays. Obviously, these mAbs bind to conformational viral epitopes because they do not react in immunoblot and by immunoprecipitation three proteins were always displayed together. The question remains open whether the mAbs react with epitopes located on one protein only or epitopes shaped by association of two or three proteins. Both the mAbs 158/5D2 and 040/4B1 reacted similarly in immunoblot and immunoprecipitation. However, in contrast to 040/4B1 the mAb 158/5D2 is able to
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Fig. 3. Chemoluminescence patterns of PEV-1 (strain Tirol) proteins precipitated by group-specific mAb 040/ 4B1 and serotype specific mAb 158/5D2. Virus was purified by ultracentrifugation on CsCl gradients and suspended in lysis buffer without SDS (A) and in lysis buffer with 1% SDS that was then removed by dialysis, respectively (B). The proteins were labeled with biotin and then precipitated by antibodies. Lane 1: rabbit antiPEV-1 serum, Lane 2: mAb 040/4B1, Lane 3: mAb 158/5D2, Lane 4: irrelevant mAb 041/3C3 (specific to another PEV serotype). Precipitates were separated by SDS-PAGE and blotted on NC membranes. M: calibration proteins. Arrows indicate specific reactions at about 37, 30 and 28 kDa, probably representing virus proteins VP1, VP2 and VP3. There is no reaction with a single protein alone.
neutralize virus. These data indicate that both mAbs recognize three-dimensional structures. It appears that mAb 040/4B1 binds to a conserved region located within the virion because of its reactivity with many serotypes and lack of neutralizing activity. Contrarily, the neutralizing mAb 158/5D2 reacts in IEM, therefore, the corresponding epitopes must be located on the outer surface. In order to diagnose PEV, the application of the group-specific mAb 040/4B1 instead of antisera will be an advantage, because antibodies to irrelevant antigens are not present. It is not possible to standardize polyclonal antisera. This procedure requires many tests with reference probes, high background staining can occur and only low dilutions are applicable. These problems may lead to wrong interpretations of results. In comparison with sera, application of these mAbs is useful for a rapid detection of group I PEV by IIF in a highly reproducible manner. Acknowledgements I am grateful to R. Ahl, P. Steinhagen and K.H. Witte who kindly supplied porcine enterovirus strains and isolates. V. Kaden carried out tests with SVDV and FMDV and H. Granzow provided the electronmicrographs for this publication. I thank Eveline Ball and
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