1 Unité de Virologie et Immunologie Moléculaires, INRA, CRJ Domaine de Vilvert, 78352 Jouy en Josas, France
2 Laboratoire d'études et de recherches en pathologie des poissons, AFSSA, BP 70, 29280 Plouzané, France
Correspondence
Michel Brémont
michel.bremont{at}jouy.inra.fr
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ABSTRACT |
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INTRODUCTION |
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To date, sleeping disease is mainly diagnosed following observation of clinical signs of fishes suspected to be infected and by the implementation of various methods such as virological assay in cell culture or histological studies (Villoing et al., 2000). The use of methods based on specific antibodies against SDV could help in the future to improve and to reduce the time needed for diagnosis. In this paper, we describe the generation and characterization of a panel of monoclonal antibodies (mAbs) directed against some of the non-structural and structural SDV proteins.
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METHODS |
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Cloning of SDV genes in pET expression vectors.
Individual SDV genes, except for nsP3, were amplified by PCR from full-length SDV genomic cDNA (C. Moriette and others, unpublished) using specific primers depicted in Table 1. pET-14b and pET-28a (Novagen) or pIVEX2.3d (Roche) prokaryotic expression vectors were used to insert the respective PCR products following appropriate restriction enzyme digestions (Table 1
). Briefly, PCR products of the nsP1, nsP4 and E2 genes were inserted into pET-14b, the nsP2 gene was inserted into pET-28a, the capsid gene was inserted into pIVEX2.3d (a pET-derived vector), while the E1 product was inserted into both pET-14b and pIVEX 2.3d vectors. The full-length SDV genomic cDNA was used to excise a 1179 nt DNA fragment, corresponding to part of the nsP3 gene, using BamHI and Bsu36I restriction enzyme sites. The ends of the DNA fragment were filled-in using the Klenow fragment of T4 DNA polymerase and inserted into the pET-28a, which had been digested with EcoRI and NotI restriction enzymes and the ends of which had been filled-in. Each plasmid construct was checked for the expression of the respective recombinant protein by T7-driven in vitro transcription/translation assay (Promega) using [35S]methionine and analysed on 8 % SDS-PAGE followed by autoradiography. Each plasmid construct was then transfected into the BL21 (DE3) Escherichia coli strain (Novagen) to overexpress the relevant SDV recombinant protein.
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DNA transfection.
EPC cell monolayers in 12-well plates (2x106 cells per well) were infected with the recombinant vaccinia virus vTF7-3 (Fuerst et al., 1986) at an m.o.i. of 5 for 1 h at 37 °C. Cell monolayers were washed twice and transfected by using the jetPEI reagent (Qbiogene) according to the supplier's instructions with individual pET plasmid constructs (0·75 µg) either encoding total recombinant protein or truncated forms of recombinant protein. Cells were incubated for 6 h at 37 °C allowing vTF7-3 to replicate, and then the temperature was shifted to 14 °C for 1216 h, the usual growth temperature for EPC cells (Fijan et al., 1983
).
Expression and purification of SDV proteins.
LuriaBertani medium (100 ml) containing appropriate antibiotic (100 µg ampicillin ml1 or 70 µg kanamycin ml1 for pET-14b and pET-28a constructs, respectively) was inoculated with 4 ml culture (OD600 of 0·6) of each recombinant E. coli clone and grown to OD600 of 0·6. The inducer IPTG was added to a final concentration of 1 mM for pET-28a clones and 0·4 mM for pET-14b clones. Cultures were further grown for either 3 or 6 h at 37 °C. Bacteria were pelleted at 3000 g for 15 min at 4 °C. Pellets were resuspended in 10 ml 50 mM Tris/HCl, 2 mM EDTA pH 8 and centrifuged for 20 min at 3000 g at 4 °C. Pellets were resuspended in 10 ml 50 mM Tris/HCl pH 8, 60 mM NaCl, and supplemented with 10 µl aprotinin, 100 µl Triton X-100 and 200 µl lysosyme (10 mg ml1). Mixtures were incubated on ice for 15 min and then Benzonase (20 µl; Merck) and 1 M MgCl2 (40 µl) were added to each sample. Samples were sonicated twice for 20 s on ice and centrifuged for 45 min at 15 000 g at 4 °C. Pellets were resuspended in 2 ml 20 mM Tris/HCl pH 8 and finally centrifuged for 15 min at 15 000 g at 4 °C. Pellets were stored at 80 °C until use.
Solubilization of the recombinant proteins.
Pellets (see above) that contain the insoluble recombinant proteins were resuspended in 0·5 M urea, incubated overnight at 4 °C under rotation and then centrifuged for 15 min at 15 000 g at 20 °C. After which most contaminant bacterial proteins are solubilized and found in the supernatant. Pellets were washed twice with 250 mM NaCl, and final pellets were resuspended in deionized water and sonicated before SDS-PAGE analysis and immunization of mice.
mAbs generation.
Six-week-old BALB/c mice were injected following different routes every 2 weeks for at least 6 weeks with either each recombinant SDV protein or 1010 p.f.u. of concentrated virus SDV. The first and second intraperitoneal injections were with complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, and the third injection by eye venous sinus was without adjuvant. Mice were bled 2 weeks later and sera were tested by immunofluorescence assay on fixed mock- and SDV-infected cells, as well as to evaluate the responsiveness of immunized mice. Positive mice received an additional boost by eye venous sinus injection and 3 days later splenocytes of mice were fused to Sp2O cells. Sera (diluted from 1 : 100 to 1 : 500) of mice injected with concentrated SDV were tested in neutralization assay (see below). Splenocytes of sera of mice that had a specific neutralizing titre were fused to Sp2O cells. Hybridomas were selected in HAT medium. Specific anti-SDV secreting hybridomas were selected by testing in immunofluorescence assay, using hybridoma supernatants against mock- and SDV-infected cells. After subcloning positive hybridomas, ascites fluids were produced in mice using standard techniques (Anderson & Potter, 1969).
Virus neutralization test.
SDV (100 p.f.u.) was incubated for 1 h at 14 °C with different dilutions (1 : 100, 1 : 250 and 1 : 500) of sera of mice injected with concentrated SDV or with serial dilutions of mAbs. As a control, SDV was incubated with culture medium. The mixtures were then used to inoculate CHSE cells in 24-well plates (5x105 cells per well) and the cells were allowed to absorb for 1 h at 14 °C. Following addition of GMEM supplemented with 2 % fetal calf serum, cells were incubated for 10 days at 10 °C and then stained with neutral red.
Immunofluorescence assay on fixed and live cells.
Cells infected with SDV or SPDV and cells transfected with each pET plasmid construct were fixed with a mixture of alcohol and acetone [1 : 1 (v/v)] at 20 °C for 15 min. Antigen detection was performed by incubation with SDV-specific mAbs diluted in PBS/Tween 20 for 45 min at room temperature. Cells were then washed, incubated with fluorescein-conjugated anti-mouse immunoglobulins (P.A.R.I.S.) for 45 min at room temperature and washed again. For assays on infected live cells, antibodies were diluted in the cell culture medium and directly used on unfixed cells following the same incubations and washing steps as described above. Cells were examined with a UV-light microscope for staining and photographed with a computer-coupled camera (Nikon).
Western blot and immunoprecipitation assays.
Reactivity of the mAbs was assessed by Western blot assays using either SDV- or SPDV-infected CHSE cell lysates, or E. coli expressed recombinant SDV proteins. Cell pellets from infected cells or overexpressed recombinant proteins were disrupted by the addition of 2x sample buffer [100 mM Tris/HCl pH 6·8, 4 % SDS, 20 % (v/v) glycerol, 0·2 % bromophenol blue, 200 mM dithiothreitol]. Approximately 2x105 cell equivalents of each infected cell extract or the 1/300 part of the recombinant proteins visualized on Fig. 1(b) were subjected to electrophoresis on 8 % SDS-PAGE, and electrotransferred onto a PVDF membrane and incubated with anti-SDV mAbs. Immunodetected antigens were visualized with horseradish peroxidase-coupled goat anti-mouse immunoglobulin (Ig) G by using an enhanced chemiluminescence detection system (ECL; Pierce).
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Immunohistochemistry (IHC) assay.
Bouin solution-fixed tissues were dehydrated through graded ethanol baths and embedded in paraffin, and then sectioned to produce slices of 56 µm thickness and mounted onto 2 % gelatin- or 10 % poly-L-lysine-coated slides. For IHC, sections were dewaxed, rehydrated, followed by blocking of endogenous peroxidase using 3 % (v/v) hydrogen peroxide. Thereafter, sections were washed in PBS and blocked in a blocking solution containing 2·5 % BSA for 10 min. Sections were incubated with primary SDV-specific mAb for 1 h at 37 °C, washed twice in PBS/Tween 20 and once in PBS, and incubated with the second antibody horseradish peroxidase-coupled goat anti-mouse IgG antibodies (P.A.R.I.S.; diluted to 1 : 200) for 30 min at 37 °C. After washes, the peroxidase substrate solution (AEC Chromogen; Sigma) was added for 10 min. Following washes in distilled water, sections were stained for 2 min with haematoxylin at room temperature and observed under the microscope.
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RESULTS |
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SDV-specific mAb generation
Non-neutralizing mAbs.
Mice were immunized using the E. coli insoluble fractions, which contain roughly 7080 % of overexpressed SDV recombinant proteins. For each immunization, insoluble fractions were thawed and sonicated to disrupt aggregates, and mice were immunized with 100 times the amount of the protein visualized in Fig. 1(b). Following screening of the hybridomas, six mAbs directed against nsP1 (labelled 8A16, E1, 19F3, I13, J4 and 71L2), two mAbs directed against nsP3 (labelled 3E17 and 26J12) and one mAb (labelled 78K5) directed against the E1 glycoprotein were selected. Some of these mAbs were further produced as ascites fluids. Because the first attempt to immunize mice against nsP2, nsP4 and E2 led to an anaphylactic shock of mice, immunizations were repeated with a new batch of the recombinant proteins that were less contaminated with bacterial proteins. To produce this new batch, the time of induction was reduced from 6 to 3 h at 37 °C, and the final pellets containing the insoluble recombinant proteins were resuspended in 6 M urea instead of 0·5 M urea, which solubilized a larger proportion of contaminant bacterial proteins leaving recombinant SDV proteins in the insoluble fraction. By using this method, we avoided anaphylactic shock of the animal, and one mAb directed against the E2 glycoprotein (labelled 51B8) was obtained and produced as ascite fluid. In contrast, even after several repeated immunizations (roughly 12 injections) over a 6 month period, we never succeeded in getting an immune response against nsP2 and nsP4 antigens.
Neutralizing mAbs.
Attempts to purify SDV free of cell contaminants failed, and thus polyethylene glycol (PEG)-concentrated SDV-infected cell supernatant was used to immunize mice. In order to select neutralizing antibodies, immunized mouse sera that had neutralizing activity were selected to fuse their splenocytes to Sp2O cells. Hybridomas were screened on SDV-infected live cells, and two neutralizing mAbs (labelled 17H23 and 40D20) were selected and amplified as ascites fluids. In addition, two non-neutralizing mAbs directed against E2 (4I16 and 49K16) were also selected.
Characterization of SDV-directed mAbs
Reactivity of the selected mAbs (summarized in Table 3) was investigated following four procedures: (i) indirect immunofluorescence (IIF) assays, (ii) Western blot assays, (iii) immunoprecipitation assays, and (iv) IHC assays. The reactivity of all the mAbs was investigated by IIF, whereas Western blot, immunoprecipitation and IHC were performed only with the mAbs produced as ascites fluids.
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Mapping of the mAbs
Each recombinant E. coli-expressed protein was analysed in four fragments. For that, initial pET SDV-derived constructs were modified in order to express a different part of each protein. Each plasmid construct was checked by analysis on SDS-PAGE of [35S]methionine labelling of in vitro transcription/translation products (data not shown). Then each construct was transfected into vTF7-3-infected EPC cells. Recognition of the expressed product by the corresponding mAb was assayed by IIF: 1A, 1B, 1C, 1D and nsP1 truncated products (Fig. 5) were assayed with anti-nsP1 mAbs (8A16, 19F3 and 71L2); fragments 3A, 3B, 3C, 3D and nsP3 truncated products were assayed with anti-nsP3 3E17 mAb; fragments E2A, E2B, E2C and E2 truncated products were assayed with anti-E2 mAbs (4I16, 49K16 and 51B8) and neutralizing 17H23 mAb; and fragments E1A, E1B, E1C, E1D and E1 truncated products were assayed with anti-E1 78K5 mAb. Results of such experiments are depicted in Fig. 5
. We succeeded in determining the regions specifically recognized by 8A16, 19F3, 71L2, 3E17, 51B8, 78K5 and 17H23 mAbs. When 4I16 and 49K16 mAbs were assayed by IIF on cells transfected with pET-E2, no fluorescence was detected and thus mapping of these two mAbs along the SDV genome was not possible.
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DISCUSSION |
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Most of the mAbs have been mapped along the SDV genome (Fig. 5). We notice that regions specifically recognized by these different mAbs are in accordance with previous observations on structures and biochemical properties of SDV proteins compared to other alphavirus proteins (Villoing, 2000
): (i) 8A16, 71L2 and 19F3 mAbs are specific for conserved regions of nsP1 not implicated in the binding of nsP1 to lipids (Ahola et al., 1999
). (ii) 3E17 mAb is specific for the conserved region of nsP3 (aa 1344). (iii) The region 201354 aa of E2 protein specifically recognized by 51B8 and 17H23 mAbs comprises the important antigenic epitopes of E2 described by Rumenapf et al. (1995)
for several alphaviruses. (iv) The 78K5 mAb is specific for a region (between aa 209 and 383) of E1 that does not comprise either the fusion domain of the protein (Levy-Mintz & Kielian, 1991
) or the transmembrane domain, which may be localized between amino acids 430 and 450 (Villoing, 2000
). The reactivity of all the mAbs was evaluated against SPDV, a virus known to be closely related to SDV (Weston et al., 2002
), and most of the mAbs cross-react with SPDV. Some other mAbs like 4I16, 49K16 and 40D20 allow discrimination between these viruses. These mAbs would be useful tools to diagnose SDV in infected trout. For example, we show here their usefulness in IHC assay. In addition, because SDV-infected fishes share the same histological lesions of fishes infected with the Infectious pancreatic necrosis virus, a birnavirus, these SDV-specific mAbs constitute a powerful tool to discriminate between IPNV- or SDV-infected trout. Little information is available on the molecular events during the replication of the salmonid alphaviruses; these mAbs will help to clarify the time course of SDV protein (non-structural and structural) synthesis in SDV-infected cells, and may also help in elucidating the kinetics of polyprotein processing during the SDV replication cycle.
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ACKNOWLEDGEMENTS |
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Received 14 March 2005;
accepted 22 July 2005.