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Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously
Journal of Virological Methods 171 (2011) 141–148
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Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously Areerat Kunanopparat a , Parin Chaivisuthangkura a , Saengchan Senapin b,c , Siwaporn Longyant a , Sombat Rukpratanporn d , Timothy W. Flegel b , Paisarn Sithigorngul a,∗ a
Department of Biology, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand CENTEX Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand d Center of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand b c
a b s t r a c t Article history: Received 19 June 2010 Received in revised form 14 October 2010 Accepted 19 October 2010 Available online 26 October 2010 Keywords: Capsid protein Immunohistochemistry Infectious myonecrosis virus (IMNV) Monoclonal antibody Penaeid shrimp Penaeus vannamei Western blot
The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CPN protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Outbreaks of disease due to infectious myonecrosis virus (IMNV) were first reported from Brazil in Pacific white shrimp Penaeus (Litopenaeus) vannamei in 2002 (Poulos et al., 2006; Pinheiro et al., 2007). The outbreaks of infection spread to Indonesia in early 2006 (Senapin et al., 2007). IMNV is an unenveloped, icosahedral virus with a diameter of 40 nm. The genome consists of a single, double-stranded RNA molecule of 7560 bp comprising two open reading frames (ORF1 and ORF2). The second half of ORF1 encodes a capsid protein with a molecular mass of 106 kDa (Poulos et al., 2006). Mortalities from IMNV infection in cultivated P. vannamei range up to 70% (Andrade et al., 2008). Apart from P. vannamei
∗ Corresponding author. Tel.: +66 2 664 1000x8511; fax: +66 2 260 0127. E-mail addresses: [email protected], paisarn [email protected] (P. Sithigorngul). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.10.015
infected naturally, two farmed shrimp species include P. monodon and P. (Litopenaeus) stylirostris were infected experimentally (Tang et al., 2005). However, no mortalities were reported for these latter two species. More recently, the wild shrimp Penaeus (Farfantepenaeus) subtilis has also reported to be susceptible to IMNV infection (Coelho et al., 2009). Gross signs of IMNV infection in P. vannamei include necrosis in muscular tissues primarily in the distal abdominal segment. These are visible as opaque, whitish, discoloration. Histological examination of tissues infection with IMNV reveals severe necrosis of muscle, fibrocytic inflammation and the appearance of cytoplasmic inclusions and lymphoid organ spheroids. However, the diagnosis of infection with IMNV based on clinical signs and histological examination is not adequate, since other pathogens such as Penaeus vannamei nodavirus (PvNV) (Tang et al., 2007) and Vibrio (Longyant et al., 2008a) and a number of other abiotic factors such as hypoxia, sudden changes in temperature or salinity can also cause muscle whitening. A highly specific and sensitive in situ
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hybridization method has been described for detection of IMNV infection in shrimp tissues with no gross signs of IMNV infection (Tang et al., 2005). Recently, more sensitive molecular methods for detection of IMNV infection were developed including RT-PCR and nested RT-PCR assays (Poulos and Lightner, 2006), real-time RT-PCR (Andrade et al., 2007) and RT-loop-mediated isothermal amplification combined with a lateral flow dipstick (Puthawibool et al., 2009). Although PCR-based methods have been used widely for detection of several shrimp viruses, they are not suitable for pond-side detection by farmers. In contrast, simpler serological based methods using monoclonal antibodies (MAbs) have been developed for detection of various shrimp pathogens such as white spot syndrome virus (WSSV) (Poulos et al., 2001; Anil et al., 2002; Chaivisuthangkura et al., 2004), Yellow head virus (YHV) (Sithigorngul et al., 2002), Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV) (Rukpratanporn et al., 2005), Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV) (Boonsanongchokying et al., 2006), Taura syndrome virus (TSV) (Longyant et al., 2008b; Chaivisuthangkura et al., 2009), Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Sithigorngul et al., 2009), Vibrio harvyei (Phainphak et al., 2005), and V. alginolyticus (Sithigorngul et al., 2006a). Although, serological based methods have lower detection sensitivity than PCR methods, they provide a field-friendly, inexpensive way to detect and confirm causative pathogens during disease outbreaks with high accuracy and in a manner (e.g., test strips) suitable for use at the pond side by unskilled personnel (Powell et al., 2006; Sithigorngul et al., 2006b, 2007a,b). Since the complete genomic sequence of IMNV is available (Tang et al., 2005), the aim of this study was to use the capsid protein of IMNV expressed heterologously to produce monoclonal antibodies for simple immuno-detection of IMNV. The ultimate aim was to use the MAb for further development of rapid immunochromatographic test strips. 2. Materials and methods 2.1. Viral preparation P. vannamei infected with IMNV naturally were obtained from a shrimp farm in the Situbondo District of East Java Province in Indonesia. They were preserved in 95% ethanol at the farm and transported to Bangkok and verified as IMNV-infected by RT-PCR (Senapin et al., 2007). The cephalothorax part of the shrimp was fixed further in Davidson’s fixative and processed for immunohistochemical analysis. Part of the abdominal muscle was homogenized in 1% Triton X-100 in PBS (0.15 M phosphate buffered saline, pH 7.2) for dot blot and Western blot assays, and part of the abdominal muscle was used for nucleic acid preparation. Uninfected P. vannamei (∼15 g) verified by RT-PCR (Senapin et al., 2007), were obtained from a farm nearby Bangkok and used as negative control in various assays. 2.2. Nucleic acid extraction Abdominal muscle from P. vannamei infected with IMNV was homogenized in lysis buffer (50 mM Tris–HCl [pH 9], 100 mM EDTA, 50 mM NaCl, 2% SDS; Flegel pers. comm.), and nucleic acid was extracted from 200 l of homogenate using a high pure viral nucleic acid kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) as described in the product manual. The extracted nucleic acid was stored at −70 ◦ C.
2.3. Cloning and expression of the IMNV capsid protein gene The IMNV coat protein gene located in the ORF1 of IMNV genome (GenBank accession number AY570982) was amplified into three parts called CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). For expression of CP-N, the primers IMNVF22 (C CAT ATG ATT GTT TCA ATG GAA AAT C) and IMNVR819 (G GAA TTC TTG TAG TGC AGT TGC TGG) with added restriction sites (underlined) were used to amplify the CP-N region by PCR with Pfx polymerase (Invitrogen, Carlbad, CA, USA) using double stranded DNA prepared by GenScript Corporation (NJ, USA) as the template. The PCR protocol consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 1 min, 68 ◦ C for 1 min and a final extension at 68 ◦ C for 15 min. The PCR product was cloned into an expression vector pTYB1 (New England, Biolabs, Hert, UK) at NdeI and EcoRI sites and transformed into E. coli strain BL21 (DE3). For expression of CP-I, primers IMNVF820 (CG GGA TCC GCT GCA AAA GAG GGT GCT CG) and IMNVR1728 (G GAA TTC TTG CAT TGA ACT CCA CGA AAA C) with added restriction sites (underlined) were used to amplify the CP-I region by RT-PCR with the Superscript Onestep RT-PCR system (Invitrogen) using IMNV cDNA as the template. For cDNA synthesis, RNA extracted from IMNV-infected shrimp was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen), an IMNVR primer (G GAA TTC TTA TAC TGT TGC TGT CGC TTG), and Escherichia coli RNaseH. The PCR condition consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 30 s, 68 ◦ C for 1 min and final extension at 68 ◦ C for 15 min. The PCR product was cloned into the expression vector pGEX-6P-1 (GE Healthcare, Björkgatan, Uppsala, Sweden) at BamHI and EcoRI sites and transformed into E. coli strain BL21. For expression of CP-C, primers IMNVF1729 (CG GGA TCC GGT AGT ATT GCA CCA GCA ATG) and IMNVR (G GAA TTC TTA TAC TGT TGC TGT CGC TTG) with added restriction sites (underlined) were used to amplify the CP-C region by PCR with Pfx polymerase (Invitrogen) using double stranded DNA, prepared by GenScript Corporation (NJ, USA) as the template. The PCR condition consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 1 min, 68 ◦ C for 1 min and final extension at 68 ◦ C for 15 min. The PCR product was cloned into an expression vector pGEX-6P-1 (GE Healthcare) at BamHI and EcoRI sites and transformed into E. coli strain BL21. The integrity of the open reading frames of the three recombinant plasmids was verified by DNA sequencing. 2.4. Preparation of recombinant CP-N, CP-I and CP-C Escherichia coli BL21 strain transformed with plasmids CPN-pTYB1, CP-I-pGEX-6P-1 or CP-C-pGEX-6P-1 was cultured in Luria–Bertani (LB) broth to the exponential phase. Expression of recombinant protein was induced with 1 mM isopropyl-d-thiogalacto-pyranoside (IPTG) for 4 h. After centrifugation at 4000 × g for 20 min at room temperature, the bacterial pellet was resuspended in a buffer containing 100 mM NaH2 PO4 , 10 mM Tris–HCl, 8 M urea pH 8, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and sonicated until a clear lysate was obtained. The lysate was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After treatment with 0.3 M KCl, recombinant fusion proteins called intein-CP-N, GST-CP-I and GST-CP-C were excised and collected in dialysis bags. Recombinant proteins were eluted with a Transblot apparatus (BioRad, Hurculis, CA, USA) at 70 V for 6 h, dialyzed and concentrated using a vacuum concentrator (Savant, Farmingdale, NY, USA). Protein concentration was determined by the Bradford assay (Bradford, 1976). The recombinant protein solutions were adjusted to 0.5 mg/ml, divided into small aliquots and stored at −70 ◦ C.
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2.5. Immunization A solution combining intein-CP-N, GST-CP-I and GST-CP-C proteins (1:1:1 mixture) with complete Freund’s adjuvant in a 1:1 ratio was injected intra-peritoneally into four Swiss mice at 0.05 mg protein per mouse. Mice were injected subsequently with the same proteins mixed with incomplete Freund’s adjuvant three more times at 2-week intervals. One week after the fourth injection, mouse antisera were collected and tested against lysates of E. coli containing intein, intein-CP-N, GST, GST-CP-I or GST-CP-C proteins by Western blotting. The most-responsive mouse was boosted subsequently 3 days before hybridoma production. The use and animal care were performed as indicated in “the guidelines on animal use protocol” under the regulation issued by the National Committee on Laboratory Animals, The National Research Council of Thailand. 2.6. Production of monoclonal antibodies A cell fusion protocol was adapted from the method developed by Köhler & Milstein (1976) with modifications described by Mosmann et al. (1979). Spleen cells (∼108 cells) were collected from the immunized mouse and fused with P3X myeloma cells (∼107 cells) with 40% polyethylene glycol. Fusion products from one mouse were plated onto 30 microculture plates (96 wells/plate) in RPMI-1640 medium supplemented with 20% fetal calf serum and 1% HAT (Gibco, Grand Island, NY, USA). After 12 days, identification of positive cultures by screening methods including dot blotting, Western blotting and immunohistochemistry were performed as described below, hybridomas were cloned by the limiting dilution method and stored in liquid nitrogen. 2.7. Specificity testing 2.7.1. Dot blotting Lysates of E. coli BL21 containing intein, intein-CP-N, GST, GSTCP-I or GST-CP-C and muscle homogenate samples (1 l/spot) from uninfected or IMNV-infected shrimp were applied to nitrocellulose membranes. These were baked at 60 ◦ C for 10 min and incubated in hybridoma conditioned medium from cultures diluted 1:20 in 1% Blotto blocking solution (1% nonfat dry milk, 0.1% Triton X-100 in PBS) for 4 h. After extensive washing in 0.1% blocking solution, the membranes were incubated in horseradish peroxidase-labeled goat anti-mouse gamma immunoglobulin heavy and light chainspecific antibody (GAM-HRP, BioRad) at 1:1000 dilution for 4 h. The membrane was then washed for 5 min in 0.1% blocking solution and incubated in a substrate mixture containing 0.03% diaminobenzidine (DAB), 0.006% hydrogen peroxide and 0.05% cobalt chloride in PBS (Sithigorngul et al., 2002). 2.7.2. Western blotting Lysates of E. coli BL21 containing intein, intein-CP-N, GST, GSTCP-I or GST-CP-C and muscle homogenate samples from uninfected or IMNV-infected shrimp were separated by 12% gel SDS-PAGE according to the method described by Laemmli (1970). Samples were electrophoresed for 3 h at 60 V and one part of the gel was stained using Coomassie brilliant blue R-250. For Western blot analysis, the samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes using a Transblot apparatus (BioRad). Nitrocellulose membranes were incubated in 1% blocking solution for 10 min and treated with MAbs or mouse anti-recombinant CP protein antiserum (preabsorbed with E. coli lysate containing intein and GST at 1:5000 dilution) for 4 h. After extensive washing in 0.1% blocking solution, the membrane was incubated with GAM-HRP at 1:1500 dilution for 4 hr. The membrane was then washed extensively as before and incubated in a substrate mixture containing 0.006% hydrogen peroxide, 0.03% DAB, 0.05% cobalt chloride in PBS.
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2.7.3. Immunohistochemistry Cephalothoraces from alcohol fixed P. vannamei specimens infected naturally with IMNV were fixed in Davidson’s fixative solution for 24 h before processing for paraffin sectioning. Serial sections (8 m thickness) of tissues were prepared and processed for indirect immuno-peroxidase staining using MAb. Peroxidase activity was visualized by incubation with 0.03% DAB and 0.006% hydrogen peroxide in PBS. Preparations were counterstained with hematoxylin and eosin y (H&E), dehydrated in graded ethanol series, cleared in xylene and mounted in Permount (Sithigorngul et al., 2002). Positive reactions were visualized as brown coloration against pink cytoplasm and purple nuclei. 2.8. MAb class and subclass determination Classes and subclasses of the mouse immunoglobulins produced by hybridomas were determined by sandwich ELISA using Mouse MonoAb ID Kit-HRP (Zymed Laboratories Research, San Francisco, CA, USA). 2.9. Cross-reactivity testing Shrimp samples infected with PemoNPV, PmDNV, PstDNV, TSV, WSSV and YHV were processed for paraffin sectioning and immunohistochemistry using MAb specific to IMNV. Results were compared to those from MAbs specific to PemoNPV (Boonsanongchokying et al., 2006), PmDNV (Rukpratanporn et al., 2005), PstDNV (Sithigorngul et al., 2009), TSV (Longyant et al., 2008b), WSSV (Chaivisuthangkura et al., 2004) and YHV (Sithigorngul et al., 2002). 2.10. Sensitivity testing with recombinant IMNV capsid proteins Purified CP-N-intein, GST-CP-I, GST-CP-C proteins were diluted serially with PBS, spotted onto nitrocellulose membranes and processed for dot blotting using the IMNV-specific MAbs generated in this study. The last dilution yielding a clear positive result was determined. 2.11. Comparison of sensitivity between MAb and one-step RT-PCR using IMNV-infected shrimp samples The sensitivity of IMNV detection in shrimp infected naturally was determined using MAbs specific to CP-N or CPC or a combination of them. The IMNV-infected shrimp samples were homogenized in 0.1% Triton-X-100 in PBS, twofold serially diluted with PBS and 1 l of each dilution was spotted onto nitrocellulose membrane and processed for dot blotting as described above. The last dilution of shrimp homogenate yielding a clear positive result was determined. The same shrimp homogenate was also diluted tenfold serially with an uninfected shrimp sample extracted with 0.1% Triton-X-100, and 1 l of each dilution was tested for IMNV by one step RT-PCR (Senapin et al., 2007). 3. Results 3.1. Capsid protein gene cloning and expression The expected PCR amplicons of 811, 924, and 1014 bp were obtained for the CP-N, CP-I and CP-C genomic regions (Fig. 1). Expression of intein-CP-N, GST-CP-I and GST-CP-C was visualized by Coomassie blue staining and the expected bands of molecular masses 84, 60 and 62 kDa, respectively, were obtained (Fig. 2A, lanes 2, 4 and 5). After the recombinant bands were cut from the gel, eluted and concentrated, highly purified fusion proteins
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A. Kunanopparat et al. / Journal of Virological Methods 171 (2011) 141–148 Table 1 MAbs specific to N and C fragments of IMNV. MAbs (isotype)
Sensitivity (fmol/spot)
Western blot
IHC
Specificity
IMN7 (IgG1) IMN12 (IgG2a) IMC1 (IgG1)
6 6 8
+++ +++ +++
+++ +++ ++
CP-N CP-N CP-C
immunoreactivity and this mouse was used for hybridoma production. Approximately 1600 hybridoma-containing wells were obtained and approximately 40 wells gave positive binding results when screened initially with combination of E. coli lysates containing CP-N-intein, GST-CP-I and GST-CP-C. Positive hybridoma clones were further screened by dot blotting and Western blotting against each E. coli lysate containing intein, GST, intein-CP-N, GST-CP-I or GST-CP-C and muscle homogenate samples from uninfected and IMNV-infected shrimp. Immunohistochemical analysis was carried out using cephalothorax sections from IMNV-infected shrimp. Ten MAbs specific to CP-N and five MAbs specific to CP-C were isolated and cloned to establish cell lines. No MAb specific to CP-I was obtained. All MAbs belonged to IgG classes. Only three MAbs, IMN7, IMN12 (specific to CP-N) and IMC1 (specific to CP-C) gave good results with various immunoassays described above (Table 1) and they were used for subsequent experiments. 3.3. Specificity of MAb
Fig. 1. Ethidium bromide stained gels of the expected PCR amplicons corresponded to IMNV capsid protein fragments: (1) CP-N (811 bp), (2) CP-I (924 bp) and (3) CP-C (1014 bp). M = DNA markers.
(2–4 mg/10 SDS-PAGE) were obtained and they were used at 0.5 mg/ml protein for immunization. 3.2. MAb production After the fourth immunization with the combination of inteinCP-N, GST-CP-I and GST-CP-C, all four mouse sera (1:5,000 dilution) reacted well to CP-N, CP-C and natural IMNV capsid protein but not with CP-I. Serum from one mouse demonstrated the strongest
All MAbs bound to a single band either recombinant protein intein-CP-N or GST-CP-C and to a band at 106 kDa in muscle homogenates from IMNV infected shrimp by Western blotting (Fig. 2C and D). Similar results were obtained by dot blotting. MAbs specific to CP-N and CP-C bound intensely to E. coli lysates containing intein-CP-N or GST-CP-C, respectively, and muscle homogenate from IMNV-infected shrimp. They did not bind to E. coli lysate containing intein, GST or GST-CP-I or to muscle homogenate from uninfected shrimp (Fig. 3). By immunohistochemical analysis, both MAbs specific to CPN and CP-C showed strong immuno-reactivity in the muscle, gill, heart, lymphoid organ, connective tissue (Fig. 4) and intertubular cells of the hepatopancreas (not shown) from IMNV-infected shrimp, similar to that observed previously by in situ hybridization (Tang et al., 2005). Therefore, immunohistochemical analysis can be used to confirm IMNV infections in a manner similar to in situ hybridization. By immunohistochemical analysis, none of the three MAbs exhibited cross-reactivity to tissues from uninfected shrimp or
Fig. 2. SDS-PAGE and Western blot analysis of antiserum and monoclonal antibody specificity. Lysates of BL21 E. coli containing (1) intein, (2) intein-CP-N, (3) GST, (4) GST-CP-I, (5) GST-CP-C and muscle homogenate from (6) uninfected or (7) IMNV infected P. vannamei were electrophoresed, then (A) stained with Coomassie brilliant blue or transferred to nitrocellulose membranes and treated with (B) mouse anti-CP-IMNV antiserum or (C) MAbs IMN12 or (D) IMC1. M = standard marker proteins. (a) Intein, (b) intein-CP-N, (c) GST, (d) GST-CP-I, (e) GST-CP-C, (f) IMNV capsid protein, and (h) hemocyanin.
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from shrimp infected with WSSV, YHV, TSV, PmDNV, PstDNV or PemoNPV (data not shown). 3.4. Sensitivity testing
Fig. 3. Dot blot analysis of MAb specificity. Lysates of BL21 Escherichia coli containing (1) intein, (2) intein-CP-N, (3) GST, (4) GST-CP-I, (5) GST-CP-C and muscle homogenate from (6) uninfected or (7) IMNV infected P. vannamei were spotted (1 l/spot) onto a nitrocellulose membrane and treated with MAbs (A) IMN12 or (B) IMC1.
Detection sensitivity of MAbs IMN7 and IMN12 determined by dot blotting using purified IM-N-intein was ca. 500 ng/ml (500 pg/spot), and this was equivalent to 6 fmol/l of antigen. Combination of MAbs IMN7 and IMN12 resulted in a 2 times increase in detection sensitivity for intein-CP-N (Fig. 5D column a), indicating that the two MAbs bound to non-overlapping epitopes. Similar results were also obtained by indirect immunoperoxidase ELISA using the combined antibodies in plates coated with purified inteinCP-N (data not shown). MAb IM-C1 showed similar detection sensitivity for purified GST-IM-C, ca. 500 ng/ml (500 pg/l), which was equivalent to
Fig. 4. Immunohistochemical analysis of MAb specificity. IMNV-infected P. vannamei tissues were treated with MAbs IMN12 (column A) or IMC1 (column C) and counterstained with eosin or (column B) stained only with hematoxylin and eosin. Strong immunoreactivities were exhibited similarly with both MAbs as brown staining in (1) muscles, (2) gills, (3) the heart, (4) connective tissue surrounding the midgut, and (5) the lymphoid organ. Pictures in rows 1–4 are the same magnification.
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Fig. 5. Sensitivity of IMNV detection by dot blotting using MAbs against CP-N and CP-C, and PCR. Lysate of (a) BL21 E. coli containing intein-CP-N, (b) GST-CP-C and (c) muscle homogenate from IMNV infected P. vannamei were serially diluted with muscle homogenate from uninfected P. vannamei and spotted onto each square of nitrocellulose membrane. The last square of each column was spotted with lysates of BL21 Escherichia coli containing (I) intein, or (G) GST or (U) muscle homogenate from uninfected P. vannamei. Each nitrocellulose membrane was treated with a single MAb or with a combination of MAbs: (A) IMN7, (B) IMN12, (C) IMC1, (D) IMN7 + IMN12 + IMC1. (E) The same sample of IMNV-infected muscle homogenate was diluted and processed for RT-PCR. *The lowest limit of detection for each method. The numbers on the left side are dilution factors of the bacterial lysates containing intein-CP-N (a) or GST-CP-C (b), and the numbers on the right side are the dilution factors of the muscular homogenate from IMNV infected P. vannamei (c).
8 fmol/spot (Table 1). It could be used to detect natural IMNV capsid protein with similar sensitivity to IMNV7 (Fig. 5C). In binding of the MAbs to natural IMNV capsid protein by dot blot assay, MAb IMN7 showed slightly stronger immunoreactivity (1:40) than IMN12 and IMC1 (Fig. 5A–C column c). Combination of the two MAbs specific to CP-N and one specific to CP-C
resulted in a 2 times increase in detection sensitivity (Fig. 5D column c). To compare the sensitivity of IMNV detection by dot blotting with that by one-step RT-PCR, the same shrimp homogenate extract used for dot blots (i.e., 0.1% Triton-X-100 extract) was used for the RT-PCR tests. A clear positive band could be observed at 1:1000
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dilution (Fig. 5E). Therefore, in comparison with one-step RT-PCR, the sensitivity of dot blotting using a combination of three MAbs was approximately 10 times less sensitive. In this case, the sensitivity of dot blotting was close to that for one step RT-PCR. Nucleic acid extraction using a commercial kit as described in Section 2.2 was not performed, since the idea was to compare the sensitivity of both methods using the same sample preparation. 4. Discussion Three MAbs specific to N and C terminal fragments of IMNV capsid protein were generated, however, MAb specific to CP-I was not obtained. Recently, MAbs specific to IMNV capsid protein were generated from recombinant His-tag IMNV corresponded to amino acid positions 300–527 of capsid protein (Seibert et al., 2010). This protein fragment overlapped with the GST-CP-I fragment. The non-responsive of mouse immune system to CP-I fragment may be due to the fact that immunogenicity of CP-I is less than that of GST, CP-N and CP-C. Since several attempts on immunization of GST-CP-I alone yielded only antibodies specific to GST but not to CP-I. All MAbs obtained from this study could be used for successful detection of IMNV infection by immunohistochemistry. The immunoreactivity results are similar to that observed previously by in situ hybridization (Tang et al., 2005). Therefore, immunohistochemical analysis can be used to confirm IMNV infections in a manner similar to in situ hybridization. Long storage of the samples in ethyl alcohol (over two years) followed by re-fixation with Davidson’s fixative before processing seemed to have no effect on the immunoreactivity in immunohistochemical analysis. Prolonged storage of shrimp tissues in Davidson fixative (10 days) did not have a deleterious effect on the in situ hybridization reaction (Andrade et al., 2008). The level of sensitivity of MAbs specific to CP-N and CP-C determined by dot blotting using purified recombinant proteins was approximately 500 pg/l similar to that previously reported for the MAbs against VP19 of WSSV (1.2–5 fmol/l; Chaivisuthangkura et al., 2010), VP28 of WSSV (500 pg/l; Anil et al., 2002, and 625 pg/l by Makesh et al., 2006), VP3 of TSV (400–800 pg/l; Longyant et al., 2008b) and capsid protein of PstDNV (300 pg/l; Sithigorngul et al., 2009). The detection limit of dot blotting using a combination of three MAbs against CP-N and CP-C was twofold higher than that of a single MAb. It is our belief that the IMNV specific MAb generated in this study can be used to develop a simple IMNV detection kit similar to the immunochromatographic strip used in WSSV and YHV detection kits, since all three MAbs bound to non-overlapping epitopes of the antigen. Acknowledgements This work was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC) Thailand to CENTEX Shrimp, Mahidol University. The authors are also indebted to farmers in Indonesia who provided IMNV infected shrimp samples. References Andrade, T.P.D., Srisuvan, T., Tang, K.F.J., Lightner, D.V., 2007. Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of infectious myonecrosis virus (IMNV). Aquaculture 264, 9–15. Andrade, T.P.D., Redman, R.M., Lightner, D.V., 2008. Evaluation of the preservation of shrimp samples with Davidson’s AFA fixative for infectious myonecrosis virus (IMNV) in situ hybridization. Aquaculture 278, 179–183. Anil, T.M., Shankar, K.M., Mohan, C.V., 2002. Monoclonal antibodies developed for sensitive detection and comparison of white spot syndrome virus isolates in India. Dis. Aquat. Organ. 51, 65–67. Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of monodon baculovirus (MBV) from shrimp. ScienceAsia 32, 371–376.
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Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chaivisuthangkura, P., Tangkhabuanbutra, J., Longyant, S., Sithigorngul, W., Rukpratanporn, S., Menasveta, P., Sithigorngul, P., 2004. Monoclonal antibodies against a truncated viral envelope protein (VP28) can detect white spot syndrome virus (WSSV) infections in shrimp. ScienceAsia 30, 359–363. Chaivisuthangkura, P., Longyant, S., Hajimasalaeh, W., Sridulyakula, P., Rukpratanporn, S., Sithigorngul, P., 2009. Improved sensitivity of Taura syndrome virus immunodetection with a monoclonal antibody against the recombinant VP2 capsid protein. J. Virol. Methods 163, 433–439. Chaivisuthangkura, P., Longyant, S., Rukpratanporn, S., Srisuk, C., Sridulyakula, P., Sithigorngul, P., 2010. Enhanced white spot syndrome virus (WSSV) detection sensitivity using monoclonal antibody specific to heterologously expressed VP19 envelope protein. Aquaculture 299, 15–20. Coelho, M.G.L., Silva, A.C.G., Nova, M.V.V., Neto, J.M.O., Lima, A.C.N., Feijo, R.G., Apolinario, D.F., Maggioni, R., Gesterira, T.C.W., 2009. Susceptibility of the wild southern brown shrimp (Farfantepenaeus subtilis) to infectious hypodermal and hematopoietic necrosis (IHHN) and infectious myonecrosis (IMN). Aquaculture 294, 1–4. Köhler, G., Milstein, C., 1976. Derivation of specific antibody producing tissue culture and tumor lines by cell fusion. Eur. J. Immunol. 6, 511–519. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 85–680. Longyant, S., Rukpratanporn, S., Chaivisuthangkura, P., Suksawad, P., Srisuk, C., Sithigorngul, W., Piyatiratitivorakul, S., Sithigorngul, P., 2008a. Identification of Vibrio spp. In vibriosis Penaeus vannamei using developed monoclonal antibodies. J. Invertebr. Pathol. 98, 63–68. Longyant, S., Poyoi, P., Chaivisuthangkura, P., Tejankura, T., Sithigorngul, W., Sithigorngul, P., Rukpratanporn, S., 2008b. Specific monoclonal antibodies raised against Taura syndrome virus (TSV) capsid protein VP3 detect TSV in single and dual infections with white spot syndrome virus (WSSV). Dis. Aquat. Organ. 79, 75–81. Makesh, M., Koteeswaran, A., Chandran, N.D.J., Manohar, B.M., Ramasamy, V., 2006. Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb. Aquaculture 261 (1), 64–71. Mosmann, T.R., Bauman, R., Williamson, A.R., 1979. Mutations affecting immunoglobulin light chain secretion by myeloma cells. I. Functional analysis by cell fusion. Eur. J. Immunol. 9, 511–516. 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Poulos, B.T., Tang, K.F.J., Pantoja, C.R., Bonami, J.R., Lightner, D.V., 2006. Purification and characterization of infectious myonecrosis virus of penaeid shrimp. J. Gen. Virol. 87, 987–996. Poulos, B.T., Lightner, D.V., 2006. Detection of infectious myonecrosis virus (IMNV) of penaeid shrimp by reverse-transcriptase polymerase chain reaction (RT-PCR). Dis. Aquat. Organ. 73, 69–72. Puthawibool, T., Senapin, S., Kiatpathomchai, W., Flegel, T.W., 2009. Detection of shrimp infectious myonecrosis virus by reveres transcription loop-mediated isothermal amplification combined with a lateral flow dipstick. J. Virol. Methods 156, 27–31. Rukpratanporn, S., Sukhumsirichart, W., Chaivisuthngkura, P., Longyant, S., Sithigorngul, W., Menasveta, P., Sithigorngul, P., 2005. Generation of monoclonal antibodies specific to hepatopancreatic parvovirus (HPV) from Penaeus monodon. Dis. Aquat. Organ. 65, 85–89. Seibert, H.C., Borsa, M., Rosa, R.D., Cargnin-Ferreira, E., Pereira, A.M.L., Grisard, E.C., Zanetti, C.R., Pinto, A.R., 2010. Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies. J. Virol. Methods 169, 169–175. Senapin, S., Phewsaiya, K., Briggs, M., Flegel, T.W., 2007. Outbreaks of infectious myonecrosis virus (IMNV) in Indonesia confirmed by genome sequencing and use of an alternative RT-PCR detection method. Aquaculture 266, 32–38. Sithigorngul, P., Rukpratanporn, S., Longyant, S., Chaivisuthangkura, P., Sithigorngul, W., Menasveta, P., 2002. Monoclonal antibodies specific to yellow-head virus (YHV) of Penaeus monodon. Dis. Aquat. Organ. 49, 71–76. Sithigorngul, W., Rengpipat, S., Tansirisittikul, A., Rukpratanporn, S., Longyant, S., Chaivisuthangkura, P., Sithigorngul, P., 2006a. Development of monoclonal antibodies for simple identification of Vibrio alginolyticus. Lett. Appl. Microbiol. 43, 436–442. 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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously Areerat Kunanopparat a , Parin Chaivisuthangkura a , Saengchan Senapin b,c , Siwaporn Longyant a , Sombat Rukpratanporn d , Timothy W. Flegel b , Paisarn Sithigorngul a,∗ a
Department of Biology, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand CENTEX Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand d Center of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand b c
a b s t r a c t Article history: Received 19 June 2010 Received in revised form 14 October 2010 Accepted 19 October 2010 Available online 26 October 2010 Keywords: Capsid protein Immunohistochemistry Infectious myonecrosis virus (IMNV) Monoclonal antibody Penaeid shrimp Penaeus vannamei Western blot
The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CPN protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Outbreaks of disease due to infectious myonecrosis virus (IMNV) were first reported from Brazil in Pacific white shrimp Penaeus (Litopenaeus) vannamei in 2002 (Poulos et al., 2006; Pinheiro et al., 2007). The outbreaks of infection spread to Indonesia in early 2006 (Senapin et al., 2007). IMNV is an unenveloped, icosahedral virus with a diameter of 40 nm. The genome consists of a single, double-stranded RNA molecule of 7560 bp comprising two open reading frames (ORF1 and ORF2). The second half of ORF1 encodes a capsid protein with a molecular mass of 106 kDa (Poulos et al., 2006). Mortalities from IMNV infection in cultivated P. vannamei range up to 70% (Andrade et al., 2008). Apart from P. vannamei
∗ Corresponding author. Tel.: +66 2 664 1000x8511; fax: +66 2 260 0127. E-mail addresses: [email protected], paisarn [email protected] (P. Sithigorngul). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.10.015
infected naturally, two farmed shrimp species include P. monodon and P. (Litopenaeus) stylirostris were infected experimentally (Tang et al., 2005). However, no mortalities were reported for these latter two species. More recently, the wild shrimp Penaeus (Farfantepenaeus) subtilis has also reported to be susceptible to IMNV infection (Coelho et al., 2009). Gross signs of IMNV infection in P. vannamei include necrosis in muscular tissues primarily in the distal abdominal segment. These are visible as opaque, whitish, discoloration. Histological examination of tissues infection with IMNV reveals severe necrosis of muscle, fibrocytic inflammation and the appearance of cytoplasmic inclusions and lymphoid organ spheroids. However, the diagnosis of infection with IMNV based on clinical signs and histological examination is not adequate, since other pathogens such as Penaeus vannamei nodavirus (PvNV) (Tang et al., 2007) and Vibrio (Longyant et al., 2008a) and a number of other abiotic factors such as hypoxia, sudden changes in temperature or salinity can also cause muscle whitening. A highly specific and sensitive in situ
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hybridization method has been described for detection of IMNV infection in shrimp tissues with no gross signs of IMNV infection (Tang et al., 2005). Recently, more sensitive molecular methods for detection of IMNV infection were developed including RT-PCR and nested RT-PCR assays (Poulos and Lightner, 2006), real-time RT-PCR (Andrade et al., 2007) and RT-loop-mediated isothermal amplification combined with a lateral flow dipstick (Puthawibool et al., 2009). Although PCR-based methods have been used widely for detection of several shrimp viruses, they are not suitable for pond-side detection by farmers. In contrast, simpler serological based methods using monoclonal antibodies (MAbs) have been developed for detection of various shrimp pathogens such as white spot syndrome virus (WSSV) (Poulos et al., 2001; Anil et al., 2002; Chaivisuthangkura et al., 2004), Yellow head virus (YHV) (Sithigorngul et al., 2002), Penaeus monodon densovirus (PmDNV) formerly called hepatopancreatic parvovirus (HPV) (Rukpratanporn et al., 2005), Penaeus monodon nucleopolyhedrovirus (PemoNPV) formerly called monodon baculovirus (MBV) (Boonsanongchokying et al., 2006), Taura syndrome virus (TSV) (Longyant et al., 2008b; Chaivisuthangkura et al., 2009), Penaeus stylirostris densovirus (PstDNV) formerly called infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Sithigorngul et al., 2009), Vibrio harvyei (Phainphak et al., 2005), and V. alginolyticus (Sithigorngul et al., 2006a). Although, serological based methods have lower detection sensitivity than PCR methods, they provide a field-friendly, inexpensive way to detect and confirm causative pathogens during disease outbreaks with high accuracy and in a manner (e.g., test strips) suitable for use at the pond side by unskilled personnel (Powell et al., 2006; Sithigorngul et al., 2006b, 2007a,b). Since the complete genomic sequence of IMNV is available (Tang et al., 2005), the aim of this study was to use the capsid protein of IMNV expressed heterologously to produce monoclonal antibodies for simple immuno-detection of IMNV. The ultimate aim was to use the MAb for further development of rapid immunochromatographic test strips. 2. Materials and methods 2.1. Viral preparation P. vannamei infected with IMNV naturally were obtained from a shrimp farm in the Situbondo District of East Java Province in Indonesia. They were preserved in 95% ethanol at the farm and transported to Bangkok and verified as IMNV-infected by RT-PCR (Senapin et al., 2007). The cephalothorax part of the shrimp was fixed further in Davidson’s fixative and processed for immunohistochemical analysis. Part of the abdominal muscle was homogenized in 1% Triton X-100 in PBS (0.15 M phosphate buffered saline, pH 7.2) for dot blot and Western blot assays, and part of the abdominal muscle was used for nucleic acid preparation. Uninfected P. vannamei (∼15 g) verified by RT-PCR (Senapin et al., 2007), were obtained from a farm nearby Bangkok and used as negative control in various assays. 2.2. Nucleic acid extraction Abdominal muscle from P. vannamei infected with IMNV was homogenized in lysis buffer (50 mM Tris–HCl [pH 9], 100 mM EDTA, 50 mM NaCl, 2% SDS; Flegel pers. comm.), and nucleic acid was extracted from 200 l of homogenate using a high pure viral nucleic acid kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) as described in the product manual. The extracted nucleic acid was stored at −70 ◦ C.
2.3. Cloning and expression of the IMNV capsid protein gene The IMNV coat protein gene located in the ORF1 of IMNV genome (GenBank accession number AY570982) was amplified into three parts called CP-N (nucleotides 2248–3045), CP-I (nucleotides 3046–3954) and CP-C (nucleotides 3955–4953). For expression of CP-N, the primers IMNVF22 (C CAT ATG ATT GTT TCA ATG GAA AAT C) and IMNVR819 (G GAA TTC TTG TAG TGC AGT TGC TGG) with added restriction sites (underlined) were used to amplify the CP-N region by PCR with Pfx polymerase (Invitrogen, Carlbad, CA, USA) using double stranded DNA prepared by GenScript Corporation (NJ, USA) as the template. The PCR protocol consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 1 min, 68 ◦ C for 1 min and a final extension at 68 ◦ C for 15 min. The PCR product was cloned into an expression vector pTYB1 (New England, Biolabs, Hert, UK) at NdeI and EcoRI sites and transformed into E. coli strain BL21 (DE3). For expression of CP-I, primers IMNVF820 (CG GGA TCC GCT GCA AAA GAG GGT GCT CG) and IMNVR1728 (G GAA TTC TTG CAT TGA ACT CCA CGA AAA C) with added restriction sites (underlined) were used to amplify the CP-I region by RT-PCR with the Superscript Onestep RT-PCR system (Invitrogen) using IMNV cDNA as the template. For cDNA synthesis, RNA extracted from IMNV-infected shrimp was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen), an IMNVR primer (G GAA TTC TTA TAC TGT TGC TGT CGC TTG), and Escherichia coli RNaseH. The PCR condition consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 30 s, 68 ◦ C for 1 min and final extension at 68 ◦ C for 15 min. The PCR product was cloned into the expression vector pGEX-6P-1 (GE Healthcare, Björkgatan, Uppsala, Sweden) at BamHI and EcoRI sites and transformed into E. coli strain BL21. For expression of CP-C, primers IMNVF1729 (CG GGA TCC GGT AGT ATT GCA CCA GCA ATG) and IMNVR (G GAA TTC TTA TAC TGT TGC TGT CGC TTG) with added restriction sites (underlined) were used to amplify the CP-C region by PCR with Pfx polymerase (Invitrogen) using double stranded DNA, prepared by GenScript Corporation (NJ, USA) as the template. The PCR condition consisted of initial denaturation at 94 ◦ C for 2 min followed by 35 cycles of 94 ◦ C for 15 s, 57 ◦ C for 1 min, 68 ◦ C for 1 min and final extension at 68 ◦ C for 15 min. The PCR product was cloned into an expression vector pGEX-6P-1 (GE Healthcare) at BamHI and EcoRI sites and transformed into E. coli strain BL21. The integrity of the open reading frames of the three recombinant plasmids was verified by DNA sequencing. 2.4. Preparation of recombinant CP-N, CP-I and CP-C Escherichia coli BL21 strain transformed with plasmids CPN-pTYB1, CP-I-pGEX-6P-1 or CP-C-pGEX-6P-1 was cultured in Luria–Bertani (LB) broth to the exponential phase. Expression of recombinant protein was induced with 1 mM isopropyl-d-thiogalacto-pyranoside (IPTG) for 4 h. After centrifugation at 4000 × g for 20 min at room temperature, the bacterial pellet was resuspended in a buffer containing 100 mM NaH2 PO4 , 10 mM Tris–HCl, 8 M urea pH 8, and 1 mM phenylmethylsulfonyl fluoride (PMSF) and sonicated until a clear lysate was obtained. The lysate was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After treatment with 0.3 M KCl, recombinant fusion proteins called intein-CP-N, GST-CP-I and GST-CP-C were excised and collected in dialysis bags. Recombinant proteins were eluted with a Transblot apparatus (BioRad, Hurculis, CA, USA) at 70 V for 6 h, dialyzed and concentrated using a vacuum concentrator (Savant, Farmingdale, NY, USA). Protein concentration was determined by the Bradford assay (Bradford, 1976). The recombinant protein solutions were adjusted to 0.5 mg/ml, divided into small aliquots and stored at −70 ◦ C.
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2.5. Immunization A solution combining intein-CP-N, GST-CP-I and GST-CP-C proteins (1:1:1 mixture) with complete Freund’s adjuvant in a 1:1 ratio was injected intra-peritoneally into four Swiss mice at 0.05 mg protein per mouse. Mice were injected subsequently with the same proteins mixed with incomplete Freund’s adjuvant three more times at 2-week intervals. One week after the fourth injection, mouse antisera were collected and tested against lysates of E. coli containing intein, intein-CP-N, GST, GST-CP-I or GST-CP-C proteins by Western blotting. The most-responsive mouse was boosted subsequently 3 days before hybridoma production. The use and animal care were performed as indicated in “the guidelines on animal use protocol” under the regulation issued by the National Committee on Laboratory Animals, The National Research Council of Thailand. 2.6. Production of monoclonal antibodies A cell fusion protocol was adapted from the method developed by Köhler & Milstein (1976) with modifications described by Mosmann et al. (1979). Spleen cells (∼108 cells) were collected from the immunized mouse and fused with P3X myeloma cells (∼107 cells) with 40% polyethylene glycol. Fusion products from one mouse were plated onto 30 microculture plates (96 wells/plate) in RPMI-1640 medium supplemented with 20% fetal calf serum and 1% HAT (Gibco, Grand Island, NY, USA). After 12 days, identification of positive cultures by screening methods including dot blotting, Western blotting and immunohistochemistry were performed as described below, hybridomas were cloned by the limiting dilution method and stored in liquid nitrogen. 2.7. Specificity testing 2.7.1. Dot blotting Lysates of E. coli BL21 containing intein, intein-CP-N, GST, GSTCP-I or GST-CP-C and muscle homogenate samples (1 l/spot) from uninfected or IMNV-infected shrimp were applied to nitrocellulose membranes. These were baked at 60 ◦ C for 10 min and incubated in hybridoma conditioned medium from cultures diluted 1:20 in 1% Blotto blocking solution (1% nonfat dry milk, 0.1% Triton X-100 in PBS) for 4 h. After extensive washing in 0.1% blocking solution, the membranes were incubated in horseradish peroxidase-labeled goat anti-mouse gamma immunoglobulin heavy and light chainspecific antibody (GAM-HRP, BioRad) at 1:1000 dilution for 4 h. The membrane was then washed for 5 min in 0.1% blocking solution and incubated in a substrate mixture containing 0.03% diaminobenzidine (DAB), 0.006% hydrogen peroxide and 0.05% cobalt chloride in PBS (Sithigorngul et al., 2002). 2.7.2. Western blotting Lysates of E. coli BL21 containing intein, intein-CP-N, GST, GSTCP-I or GST-CP-C and muscle homogenate samples from uninfected or IMNV-infected shrimp were separated by 12% gel SDS-PAGE according to the method described by Laemmli (1970). Samples were electrophoresed for 3 h at 60 V and one part of the gel was stained using Coomassie brilliant blue R-250. For Western blot analysis, the samples resolved by SDS-PAGE were transferred onto nitrocellulose membranes using a Transblot apparatus (BioRad). Nitrocellulose membranes were incubated in 1% blocking solution for 10 min and treated with MAbs or mouse anti-recombinant CP protein antiserum (preabsorbed with E. coli lysate containing intein and GST at 1:5000 dilution) for 4 h. After extensive washing in 0.1% blocking solution, the membrane was incubated with GAM-HRP at 1:1500 dilution for 4 hr. The membrane was then washed extensively as before and incubated in a substrate mixture containing 0.006% hydrogen peroxide, 0.03% DAB, 0.05% cobalt chloride in PBS.
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2.7.3. Immunohistochemistry Cephalothoraces from alcohol fixed P. vannamei specimens infected naturally with IMNV were fixed in Davidson’s fixative solution for 24 h before processing for paraffin sectioning. Serial sections (8 m thickness) of tissues were prepared and processed for indirect immuno-peroxidase staining using MAb. Peroxidase activity was visualized by incubation with 0.03% DAB and 0.006% hydrogen peroxide in PBS. Preparations were counterstained with hematoxylin and eosin y (H&E), dehydrated in graded ethanol series, cleared in xylene and mounted in Permount (Sithigorngul et al., 2002). Positive reactions were visualized as brown coloration against pink cytoplasm and purple nuclei. 2.8. MAb class and subclass determination Classes and subclasses of the mouse immunoglobulins produced by hybridomas were determined by sandwich ELISA using Mouse MonoAb ID Kit-HRP (Zymed Laboratories Research, San Francisco, CA, USA). 2.9. Cross-reactivity testing Shrimp samples infected with PemoNPV, PmDNV, PstDNV, TSV, WSSV and YHV were processed for paraffin sectioning and immunohistochemistry using MAb specific to IMNV. Results were compared to those from MAbs specific to PemoNPV (Boonsanongchokying et al., 2006), PmDNV (Rukpratanporn et al., 2005), PstDNV (Sithigorngul et al., 2009), TSV (Longyant et al., 2008b), WSSV (Chaivisuthangkura et al., 2004) and YHV (Sithigorngul et al., 2002). 2.10. Sensitivity testing with recombinant IMNV capsid proteins Purified CP-N-intein, GST-CP-I, GST-CP-C proteins were diluted serially with PBS, spotted onto nitrocellulose membranes and processed for dot blotting using the IMNV-specific MAbs generated in this study. The last dilution yielding a clear positive result was determined. 2.11. Comparison of sensitivity between MAb and one-step RT-PCR using IMNV-infected shrimp samples The sensitivity of IMNV detection in shrimp infected naturally was determined using MAbs specific to CP-N or CPC or a combination of them. The IMNV-infected shrimp samples were homogenized in 0.1% Triton-X-100 in PBS, twofold serially diluted with PBS and 1 l of each dilution was spotted onto nitrocellulose membrane and processed for dot blotting as described above. The last dilution of shrimp homogenate yielding a clear positive result was determined. The same shrimp homogenate was also diluted tenfold serially with an uninfected shrimp sample extracted with 0.1% Triton-X-100, and 1 l of each dilution was tested for IMNV by one step RT-PCR (Senapin et al., 2007). 3. Results 3.1. Capsid protein gene cloning and expression The expected PCR amplicons of 811, 924, and 1014 bp were obtained for the CP-N, CP-I and CP-C genomic regions (Fig. 1). Expression of intein-CP-N, GST-CP-I and GST-CP-C was visualized by Coomassie blue staining and the expected bands of molecular masses 84, 60 and 62 kDa, respectively, were obtained (Fig. 2A, lanes 2, 4 and 5). After the recombinant bands were cut from the gel, eluted and concentrated, highly purified fusion proteins
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A. Kunanopparat et al. / Journal of Virological Methods 171 (2011) 141–148 Table 1 MAbs specific to N and C fragments of IMNV. MAbs (isotype)
Sensitivity (fmol/spot)
Western blot
IHC
Specificity
IMN7 (IgG1) IMN12 (IgG2a) IMC1 (IgG1)
6 6 8
+++ +++ +++
+++ +++ ++
CP-N CP-N CP-C
immunoreactivity and this mouse was used for hybridoma production. Approximately 1600 hybridoma-containing wells were obtained and approximately 40 wells gave positive binding results when screened initially with combination of E. coli lysates containing CP-N-intein, GST-CP-I and GST-CP-C. Positive hybridoma clones were further screened by dot blotting and Western blotting against each E. coli lysate containing intein, GST, intein-CP-N, GST-CP-I or GST-CP-C and muscle homogenate samples from uninfected and IMNV-infected shrimp. Immunohistochemical analysis was carried out using cephalothorax sections from IMNV-infected shrimp. Ten MAbs specific to CP-N and five MAbs specific to CP-C were isolated and cloned to establish cell lines. No MAb specific to CP-I was obtained. All MAbs belonged to IgG classes. Only three MAbs, IMN7, IMN12 (specific to CP-N) and IMC1 (specific to CP-C) gave good results with various immunoassays described above (Table 1) and they were used for subsequent experiments. 3.3. Specificity of MAb
Fig. 1. Ethidium bromide stained gels of the expected PCR amplicons corresponded to IMNV capsid protein fragments: (1) CP-N (811 bp), (2) CP-I (924 bp) and (3) CP-C (1014 bp). M = DNA markers.
(2–4 mg/10 SDS-PAGE) were obtained and they were used at 0.5 mg/ml protein for immunization. 3.2. MAb production After the fourth immunization with the combination of inteinCP-N, GST-CP-I and GST-CP-C, all four mouse sera (1:5,000 dilution) reacted well to CP-N, CP-C and natural IMNV capsid protein but not with CP-I. Serum from one mouse demonstrated the strongest
All MAbs bound to a single band either recombinant protein intein-CP-N or GST-CP-C and to a band at 106 kDa in muscle homogenates from IMNV infected shrimp by Western blotting (Fig. 2C and D). Similar results were obtained by dot blotting. MAbs specific to CP-N and CP-C bound intensely to E. coli lysates containing intein-CP-N or GST-CP-C, respectively, and muscle homogenate from IMNV-infected shrimp. They did not bind to E. coli lysate containing intein, GST or GST-CP-I or to muscle homogenate from uninfected shrimp (Fig. 3). By immunohistochemical analysis, both MAbs specific to CPN and CP-C showed strong immuno-reactivity in the muscle, gill, heart, lymphoid organ, connective tissue (Fig. 4) and intertubular cells of the hepatopancreas (not shown) from IMNV-infected shrimp, similar to that observed previously by in situ hybridization (Tang et al., 2005). Therefore, immunohistochemical analysis can be used to confirm IMNV infections in a manner similar to in situ hybridization. By immunohistochemical analysis, none of the three MAbs exhibited cross-reactivity to tissues from uninfected shrimp or
Fig. 2. SDS-PAGE and Western blot analysis of antiserum and monoclonal antibody specificity. Lysates of BL21 E. coli containing (1) intein, (2) intein-CP-N, (3) GST, (4) GST-CP-I, (5) GST-CP-C and muscle homogenate from (6) uninfected or (7) IMNV infected P. vannamei were electrophoresed, then (A) stained with Coomassie brilliant blue or transferred to nitrocellulose membranes and treated with (B) mouse anti-CP-IMNV antiserum or (C) MAbs IMN12 or (D) IMC1. M = standard marker proteins. (a) Intein, (b) intein-CP-N, (c) GST, (d) GST-CP-I, (e) GST-CP-C, (f) IMNV capsid protein, and (h) hemocyanin.
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from shrimp infected with WSSV, YHV, TSV, PmDNV, PstDNV or PemoNPV (data not shown). 3.4. Sensitivity testing
Fig. 3. Dot blot analysis of MAb specificity. Lysates of BL21 Escherichia coli containing (1) intein, (2) intein-CP-N, (3) GST, (4) GST-CP-I, (5) GST-CP-C and muscle homogenate from (6) uninfected or (7) IMNV infected P. vannamei were spotted (1 l/spot) onto a nitrocellulose membrane and treated with MAbs (A) IMN12 or (B) IMC1.
Detection sensitivity of MAbs IMN7 and IMN12 determined by dot blotting using purified IM-N-intein was ca. 500 ng/ml (500 pg/spot), and this was equivalent to 6 fmol/l of antigen. Combination of MAbs IMN7 and IMN12 resulted in a 2 times increase in detection sensitivity for intein-CP-N (Fig. 5D column a), indicating that the two MAbs bound to non-overlapping epitopes. Similar results were also obtained by indirect immunoperoxidase ELISA using the combined antibodies in plates coated with purified inteinCP-N (data not shown). MAb IM-C1 showed similar detection sensitivity for purified GST-IM-C, ca. 500 ng/ml (500 pg/l), which was equivalent to
Fig. 4. Immunohistochemical analysis of MAb specificity. IMNV-infected P. vannamei tissues were treated with MAbs IMN12 (column A) or IMC1 (column C) and counterstained with eosin or (column B) stained only with hematoxylin and eosin. Strong immunoreactivities were exhibited similarly with both MAbs as brown staining in (1) muscles, (2) gills, (3) the heart, (4) connective tissue surrounding the midgut, and (5) the lymphoid organ. Pictures in rows 1–4 are the same magnification.
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Fig. 5. Sensitivity of IMNV detection by dot blotting using MAbs against CP-N and CP-C, and PCR. Lysate of (a) BL21 E. coli containing intein-CP-N, (b) GST-CP-C and (c) muscle homogenate from IMNV infected P. vannamei were serially diluted with muscle homogenate from uninfected P. vannamei and spotted onto each square of nitrocellulose membrane. The last square of each column was spotted with lysates of BL21 Escherichia coli containing (I) intein, or (G) GST or (U) muscle homogenate from uninfected P. vannamei. Each nitrocellulose membrane was treated with a single MAb or with a combination of MAbs: (A) IMN7, (B) IMN12, (C) IMC1, (D) IMN7 + IMN12 + IMC1. (E) The same sample of IMNV-infected muscle homogenate was diluted and processed for RT-PCR. *The lowest limit of detection for each method. The numbers on the left side are dilution factors of the bacterial lysates containing intein-CP-N (a) or GST-CP-C (b), and the numbers on the right side are the dilution factors of the muscular homogenate from IMNV infected P. vannamei (c).
8 fmol/spot (Table 1). It could be used to detect natural IMNV capsid protein with similar sensitivity to IMNV7 (Fig. 5C). In binding of the MAbs to natural IMNV capsid protein by dot blot assay, MAb IMN7 showed slightly stronger immunoreactivity (1:40) than IMN12 and IMC1 (Fig. 5A–C column c). Combination of the two MAbs specific to CP-N and one specific to CP-C
resulted in a 2 times increase in detection sensitivity (Fig. 5D column c). To compare the sensitivity of IMNV detection by dot blotting with that by one-step RT-PCR, the same shrimp homogenate extract used for dot blots (i.e., 0.1% Triton-X-100 extract) was used for the RT-PCR tests. A clear positive band could be observed at 1:1000
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dilution (Fig. 5E). Therefore, in comparison with one-step RT-PCR, the sensitivity of dot blotting using a combination of three MAbs was approximately 10 times less sensitive. In this case, the sensitivity of dot blotting was close to that for one step RT-PCR. Nucleic acid extraction using a commercial kit as described in Section 2.2 was not performed, since the idea was to compare the sensitivity of both methods using the same sample preparation. 4. Discussion Three MAbs specific to N and C terminal fragments of IMNV capsid protein were generated, however, MAb specific to CP-I was not obtained. Recently, MAbs specific to IMNV capsid protein were generated from recombinant His-tag IMNV corresponded to amino acid positions 300–527 of capsid protein (Seibert et al., 2010). This protein fragment overlapped with the GST-CP-I fragment. The non-responsive of mouse immune system to CP-I fragment may be due to the fact that immunogenicity of CP-I is less than that of GST, CP-N and CP-C. Since several attempts on immunization of GST-CP-I alone yielded only antibodies specific to GST but not to CP-I. All MAbs obtained from this study could be used for successful detection of IMNV infection by immunohistochemistry. The immunoreactivity results are similar to that observed previously by in situ hybridization (Tang et al., 2005). Therefore, immunohistochemical analysis can be used to confirm IMNV infections in a manner similar to in situ hybridization. Long storage of the samples in ethyl alcohol (over two years) followed by re-fixation with Davidson’s fixative before processing seemed to have no effect on the immunoreactivity in immunohistochemical analysis. Prolonged storage of shrimp tissues in Davidson fixative (10 days) did not have a deleterious effect on the in situ hybridization reaction (Andrade et al., 2008). The level of sensitivity of MAbs specific to CP-N and CP-C determined by dot blotting using purified recombinant proteins was approximately 500 pg/l similar to that previously reported for the MAbs against VP19 of WSSV (1.2–5 fmol/l; Chaivisuthangkura et al., 2010), VP28 of WSSV (500 pg/l; Anil et al., 2002, and 625 pg/l by Makesh et al., 2006), VP3 of TSV (400–800 pg/l; Longyant et al., 2008b) and capsid protein of PstDNV (300 pg/l; Sithigorngul et al., 2009). The detection limit of dot blotting using a combination of three MAbs against CP-N and CP-C was twofold higher than that of a single MAb. It is our belief that the IMNV specific MAb generated in this study can be used to develop a simple IMNV detection kit similar to the immunochromatographic strip used in WSSV and YHV detection kits, since all three MAbs bound to non-overlapping epitopes of the antigen. Acknowledgements This work was supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC) Thailand to CENTEX Shrimp, Mahidol University. The authors are also indebted to farmers in Indonesia who provided IMNV infected shrimp samples. References Andrade, T.P.D., Srisuvan, T., Tang, K.F.J., Lightner, D.V., 2007. Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of infectious myonecrosis virus (IMNV). Aquaculture 264, 9–15. Andrade, T.P.D., Redman, R.M., Lightner, D.V., 2008. Evaluation of the preservation of shrimp samples with Davidson’s AFA fixative for infectious myonecrosis virus (IMNV) in situ hybridization. Aquaculture 278, 179–183. Anil, T.M., Shankar, K.M., Mohan, C.V., 2002. Monoclonal antibodies developed for sensitive detection and comparison of white spot syndrome virus isolates in India. Dis. Aquat. Organ. 51, 65–67. Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of monodon baculovirus (MBV) from shrimp. ScienceAsia 32, 371–376.
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