doi:10.1111/jfd.12230
Journal of Fish Diseases 2015, 38, 249–258
Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins S-L Hu1, C-J Liou2, Y-H Cheng1, J-C Yiu3, P P Chiou4 and Y-S Lai1 1 2 3 4
Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan Department of Horticulture, National Ilan University, Yilan, Taiwan Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Yilan, Taiwan
Abstract
Grouper iridovirus (GIV) is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. The study of GIV pathogenicity has been hampered by the lack of proper immunological reagents to study the expression and function of viral proteins in the infected cells. In this study, two mouse monoclonal antibodies (mAbs) against GIV 55L and 97L proteins were produced. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen these hybridomas, resulting in the identification of two high-affinity mAbs named GIV55L-mAb-2 and GIV97L-mAb-3, respectively. Both mAbs belong to the IgG1 isotype and were effective in detecting their respective target viral protein. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analyses of GIV-infected GK cells revealed that GIV 97L is an immediate early gene, whereas GIV 55L a late one. The localization of 55L and 97L in GIV-infected cells was further characterized by immunofluorescence microscopy with the mAbs. The 55L protein mainly aggregated in the cytoplasm while 97L distributed in both the nucleus and cytoplasm of the infected cells. These studies demonstrate the validity of the two mAbs as immunodiagnostic and research reagents. Correspondence Y-S Lai, Department of Biotechnology and Animal Science, National Ilan University, 1, Sec. 1, Shen-Lung Road, Yilan 26047, Taiwan (e-mail: [email protected]) and P P Chiou, Marine Research Station, Institute of Cellular & Organismic Biology, Academia Sinica, 23-10 Dawun Road, Jiaoxi, Yilan 26242, Taiwan (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Keywords: ELISA, grouper iridovirus, immunofluorescence, monoclonal antibodies.
Introduction
Grouper (Epinephelus spp) is one of the most economically important cultured marine fish in many Asian countries. A major constraint in grouper hatchery production and farming is infectious disease (Chi 1997; Lai et al. 2001a,b). Iridovirus is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages (Lai et al. 2000, 2003). Iridoviruses are large, icosahedral, double-stranded DNA virus with a viral particle size ranging from 120 to 350 nm in diameter (Williams 1996; Chinchar et al. 2005). The iridoviridae family comprises five genera: iridovirus, chloriridovirus, ranavirus, lymphocystivirus and megalocytivirus, whose members can infect invertebrates and vertebrates (Williams et al. 2000; Williams, Barbosa-Solomieu & Chinchar 2005; Eaton et al. 2007). Vertebrate iridoviruses have been reported in fish, amphibians and reptiles, and many of them can lead to severe systemic diseases in their hosts (Williams 1996). Grouper iridovirus (GIV), originally isolated from southern Taiwan, is a member of the ranavirus genus and has resulted in significant economic losses in the grouper aquaculture industry (Lai et al. 2000; Murali et al. 2002). The genome of GIV consists of 139 793 bp in length with a 49% G + C content. The genome is predicted to encode 120 open reading frames (ORFs), whose sizes ranging from 62 to 1268 amino acids (Tsai
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et al. 2005). Despite the devastating impact of iridoviruses on the grouper aquaculture industry, the study on the mechanism of iridoviral pathogenicity remains difficult due to the lack of proper tools. In an earlier investigation, we established 7 GIV-susceptible cell lines from yellow grouper, Epinephelus awoara (Lai et al. 2003) and two cell lines from Barramundi, Lates calcarifer, in which GIV was capable of inducing apoptosis (Lai et al. 2008). These cell lines have been shown to be valuable tools to assist in the investigation on the cell death caused by GIV (Chiou, Chen & Lai 2009; Pham et al. 2012). Yet, other tools such as immunological reagents are still in need to assist in the study of the pathogenicity mechanism of iridoviruses. Monoclonal antibodies (mAbs) are efficient tools for detection, screening and characterization of biomolecules and have wide applications in disease diagnosis and immunotherapy (Shepard et al. 1991; Toi et al. 2004). In virus research, specific mAbs are widely used in diagnosis, virus pathogenesis and immunotherapy of viral disease (Shi et al. 2003; Fofana et al. 2013; Wang et al. 2013; Zhang et al. 2013a,b). The aim of this study was to produce mAbs that could assist in the immunological detection of GIV infection. We showed here the development and characterization of two mAbs against GIV 55L and 97L. These two mAbs were specific to their target antigens, and their ability to serve as detection tools for GIV infection was demonstrated by enzymelinked immunosorbent assay (ELISA), immunoblotting and immunofluorescence analyses.
Materials and methods
Virus and cells GK (grouper kidney) cells (Lai et al. 2000) were grown at 28 °C in Leibovitz’s L15 medium supplemented with 10% foetal bovine serum (FBS), 100 IU mL 1 penicillin and 100 lg mL 1 streptomycin. The propagation and purification of GIV was conducted in GK cells as described previously (Lai et al. 2000). Construction of expression plasmids The complete ORFs of GIV 55L and GIV 97L were amplified by PCR from GIV genomic DNA, using oligonucleotide primers containing designed restriction enzyme cleavage sites. The following Ó 2014 John Wiley & Sons Ltd
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primers were used for GIV 55L: forward5′-CGCGGATCCATGGATCAGTGGCTG-3′ (BamH Ι site underlined), and reverse-5′-CCG CTCGAGTTTAGTCTCCATCAA-3′ (Xho Ι site underlined). PCR was carried out under the following conditions: 10 min at 95 °C for 1 cycle; 1 min at 95 °C, 1 min at 56 °C and 1 min at 72 °C, for 35 cycles; 10 min at 72 °C for 1 cycle. The following primers were used for GIV 097L: forward-5′-CGCGGATCCATGGAATGTTTATA C-3′ (BamH Ι site underlined), reverse-5′-CCGC TCGAGCACAAACCCAAATTT-3′ (Xho Ι site underlined). PCR was carried out under the following conditions: 5 min at 95 °C for 1 cycle; 30 s at 95 °C, 40 s at 55 °C and 1 min at 72 °C, for 35 cycles; 10 min at 72 °C for 1 cycle. The amplified fragment of GIV 55L was cloned into the pGEX-6P-1 expression vector and GIV 97L into the pET23a expression vector. These constructs were transformed into E.coli XL1-Blue, and the transformation was confirmed by restriction enzyme digestion and DNA sequencing. Expression and purification of recombinant proteins After transformation into BL21 (DE3) cells, a single positive clone was inoculated in 3 mL of Luria– Bertani (LB) broth containing 100 lg mL 1 ampicillin and cultured overnight at 37 °C. The overnight culture of E. coli cells was inoculated into 500 mL LB supplemented with 100 lg mL 1 of ampicillin. When the OD600 reached 0.5, IPTG was added at a final concentration of 1 mM to induce protein expression for 24 h. The cells (pET23a-97L) were harvested by centrifugation (4000 g, 20 min), and the pellet was resuspended in lysis buffer A (6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0; 5 mL buffer A/1 g cell pellet) overnight at 4 °C. The cells were disrupted by lysis buffer A, and the insoluble debris was removed by centrifugation at 10 000 g for 30 min. The supernatant was then applied directly onto a Ni-NTA agarose affinity chromatography column and washed sequentially by lysis buffer A containing 1, 5, 10, 20 and 200 mM imidazole. The elute was collected in 3-mL fractions and analysed by SDS-PAGE. The fractions containing protein of the expected molecular weight were pooled and dialysed with S100 buffer (25 mM HEPES, 20% Glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT, pH 7.9). The
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pGEX-6p-1-55L recombinant proteins were purified using GST-agarose (Fluka) following the manufacturer’s protocol. After dialysis, the concentration of purified recombinant protein was measured using a Bradford assay. Hybridoma preparation BALB/c mice (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used for immunization. Each mouse (8 weeks old) was injected intraperitoneally with 100 lg of recombinant protein, which was emulsified with an equal volume of Freund’s complete adjuvant. After 14 days, a booster injection of the same dosage, but prepared in Freund’s incomplete adjuvant, was given as described above. Two more injections were administered at 2-week intervals. Two days after the last injection, spleen cells were removed and fused with mouse myeloma cells (NS1) in the presence of 50% polyethylene glycol (MW 1500). The fused cells were then resuspended in RPMI1640 medium supplemented with 10% FBS, 19 hypoxanthine–aminopterin–thymidine (HAT media supplement (509) Hybri-MaxTM, Sigma). Seven to ten days after fusion, the culture medium from hybridoma cells was screened for the presence of specific antibodies by ELISA. Cells in the positive wells were further subcloned by limiting dilution and screened by enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assay ELISA plates (Nunc) were coated with 100 lL of antigen protein suspension (1 ng lL 1) per well diluted in PBS containing 0.05% NaN3 and were incubated at 4 °C overnight. The plates were then blocked with 5% skim milk in PBS for 2 h at 37 °C and washed three times with PBST [PBS containing 0.05% (v/v) Tween-20]. One hundred microlitres of mouse serum and hybridoma supernatant diluted in PBST was added to each well. After 2-h incubation at 37 °C, plates were rinsed three times with PBST, and 100 lL of 1:5000-diluted horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (Santa Cruz Biotechnology) was added to each well. The plates were incubated for 1 h at 37 °C. After washing the plate three times with PBST, the enzyme activity was determined by adding TMB single solution substrate (Invitrogen). After 30-min Ó 2014 John Wiley & Sons Ltd
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incubation in dark, the light absorbance of each well was read at 650 nm with an ELISA plate reader (SpectraMax M2, Molecular Devices). Mouse mAb Isotype determination and purification The isotyping of the established mAbs was conducted by ELISA with a mouse mAb isotyping kit (Sigma). Each BALB/c mice (20 weeks old) was injected intraperitoneally with 200-lL pristine, and 10 days later, 5 9 106 hybridoma cells would be injected into the mouse. The ascitic fluids were subsequently collected for mAbs purification using Protein G-agarose (Thermo, NAbTM protein G spin kit) following the manufacturer’s protocol. The animal experiments and ascites production were conducted by following the Institutional Animal Care and Use Committee (IACUC) guidelines of the National Laboratory Animal Center (Taiwan). Reverse-transcription polymerase chain reaction (RT-PCR) GK cells were infected with GIV at an MOI of 5 and harvested at 0, 1, 3, 6, 12 and 24 h postinfection (hpi) in the presence or absence of cycloheximide (CHX; 50 g mL 1; Sigma Aldrich, St. Louis, MO). The total RNA was extracted using TRIzol reagent (Invitrogen) with RNase-free DNase (New England Biolabs) to remove contaminated DNA. Two micrograms of the total RNA was subsequently reverse-transcribed into the firststranded cDNA using reverse transcriptase (Roche) with random primers, following the manufacturer’s protocol. PCR was carried out under the following conditions: 2 lL of cDNA, 2 lM forward primer, 2 lM reverse primer, 2.5 lM of each dNTP, 19 PCR buffer and 2.5 U Tag DNA polymerase (Viogene) to a final volume of 50 lL. After amplification, the PCR products were electrophoresed in a 1.2% agarose-TAE buffer gel and stained with ethidium bromide. Western blotting Western blotting was performed with lysates collected from GIV-infected GK cells at an MOI of 5 in the presence or absence of cytosine arabinoside (AraC; 40 lg mL 1; Sigma Aldrich). At 1 h, 3 h, 6 h, 12 h and 24 hpi, GIV-infected or
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mock-infected cells were rinsed with PBS and lysed in 29 SDS/sample buffer (100 mM Tris– HCl (pH 6.8), 10% b-mercaptoethanol, 4% SDS, 0.2% bromophenol blue and 20% glycerol). Cell lysates were subjected to 12% SDS-PAGE (120 V, 1.5 h) and transferred onto PVDF membrane (Pall Corporation) at 400 mA for 2 h in a Bio-Rad mini Trans-Blot electrophoretic transfer cell for Western blotting analysis. The membrane was washed in PBST and blocked with 5% skimmed milk overnight at 4 °C. After washing, the membrane was reacted with GIV55L-mAb-2 (500 lg mL 1, 1:5000), GIV97L-mAb-3 (500 lg mL 1, 1:5000), GIV45R (MCP) (mouse serum, 1:5000) or b-actin (3.1 mg mL 1, 1:5000) (Sigma, catalog A5441) for 2 h at 37 °C, washed again and then incubated with alkaline phosphatase-conjugated goat anti-mouse immunoglobulins (400 lg mL 1, 1:5000) (catalog 2058; Santa Cruz Biotechnology) at 37 °C for 1 h. Specific bands were visualized with 5-bromo-4chloro-3-indolyl phosphate (BCIP) and 4-nitroblue tetrazolium chloride (NBT). Immunofluorescent microscopy GK cells grown on Lab-Tek chamber slideTM (four-well Permanox Slide, Nalge Nunc International Corp.) were infected with GIV at an MOI of 5. At 1, 6 and 12 hpi, the culture media were removed and cells were washed with PBS. The cells were then fixed in 2% para-formaldehyde/ 0.1% Triton X-100 for 30 min on ice. After removal of fixative, the cells were washed twice in ice-cold PBS and incubated with designated specific mAb (500 lg mL 1, 1:100) (GIV55L-mAb2 or GIV97L-mAb-3) at 4 °C for 60 min. After wash with ice-cold PBS three times, the fluorescein-labelled goat anti-mouse IgG (H + L) (500 lg mL 1, 1:50) (KPL catalog 02-18-06) was added into the chamber slide and incubated at 4 °C for 60 min. The slides were washed with ice-cold PBS three times, mounted and subjected to examination under a fluorescence microscope (Axio Observer, Zeiss).
Results
Sequence analysis of GIV 55L and 97L GIV 55L (GenBank accession no. AAV91073) is 1128 bp in length, encoding a putative protein of Ó 2014 John Wiley & Sons Ltd
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376 amino acids with a predicted molecular mass of 41.57 kDa. The deduced amino acid sequence of GIV 55L is 99% identical to the homologs of Singapore grouper iridovirus (SGIV) (AAS18099) and 47% to frog virus 3 (FV3) (AAT09740), Ambystoma tigrinum virus (ATV) (AAP33202) and tiger frog virus (TFV) (AAL77809) (Fig. 1a). The predicated protein structure of GIV55L and the homologs of other iridoviruses suggest that it is an RNAse III (Fig. 1). GIV 97L (GenBank accession no. AAV91107) is of 975 bp and encodes a putative protein of 325 amino acids with a predicted molecular mass of 36.68 kDa. The deduced amino acid sequence of GIV 97L is 98, 45, 44 and 44% identical to the homologs of SGIV (AAS18161), FV3 (AAT09733), ATV (AA P33208) and TFV (AAL77808), respectively (Fig. 1b). Expression and purification of recombinant GIV 55L and 97L proteins Plasmids pGEX-6P-1-55L and pET23a-97L were constructed to express recombinant GST-55L and His-97L proteins, respectively. The estimated size of recombinant GST-55L fusion protein was 67.57 kDa, and that of His-97L, 36.68 kDa. As shown in Fig. 2, at 24 h of induction with 1 mM IPTG, the two recombinant proteins were successfully expressed, as demonstrated by the presence of bands of the respective estimated size. Production and characterization of GIV 55L and 97L mAbs To produce GIV 55L and 97L monoclonal antibodies, NS1 myeloma cells were fused with spleen cells removed from four mice immunized with purified recombinant GST-55L and His-97L proteins, respectively. The fused cells were subsequently screened by ELISA against recombinant 55L and 97L. Six positive clones (GIV55L-mAb1, GIV55L-mAb-2, GIV55L-mAb-3, GIV55LmAb-4, GIV55L-mAb-5 and GIV55L-mAb-6) were identified against GIV 55 of 52 clones, and 8 positive clones (GIV97L-mAb-1, GIV97L-mAb2, GIV97L-mAb-3, GIV97L-mAb-4, GIV97LmAb-5, GIV97L-mAb-6, GIV97L-mAb-7 and GIV97L-mAb-8) against GIV 97L identified of 85. During the screening process, the strategy of limiting dilution was adopted and repeated three times. By ELISA analysis, clones GIV55L-mAb-2
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Figure 1 Multiple amino acid sequence alignment of 55L and 97L of GIV with related genes sequences of four other iridoviruses. The highly conserved residues in all sequences are marked by a pound symbol under the alignment. (a) GIV 55L amino acid sequence alignment. (b) GIV 97L amino acid sequence alignment.
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Figure 2 Purification of recombinant GIV 55L and 97L proteins. Recombinant proteins were harvested from transformed E. coli, and the proteins were further purified by GST or Ni-NTA Agarose affinity chromatography column. (a) Recombinant GST-55L: lane 1, prestained protein marker; lane 2, E. coli BL21(DE3)-pGEX-6p-1-55L; lane 3, E coli BL21 (DE3)-pGEX-6p-1-55L/IPTG; lane 4, purified recombinant GST-55L. (b) Recombinant 97L: lane 1, prestained protein marker; lane 2, E. coli BL21(DE3)-pET23a-97L; lane 3, E. coli BL21(DE3)-pET23a-97L/IPTG; lane 4, purified recombinant His-97L.
and GIV97L-mAb-3 showed the greatest reactivity against 55L and 97L, respectively. Thus, the GIV55L-mAb-2 and GIV97L-mAb-3 hybridoma cells were injected into mice, from which 55L- and 97L-specific mAbs were successfully produced (data not showed). Further isotyping showed that both 55L and 97L mAbs were of IgG1 isotype (data not showed). Expression pattern of 55L and 97L over the course of the GIV infection Experiments were carried out to verify the feasibility of the two newly established mAbs as immunological reagents for detecting 55L and 97L in GIV-infected cells. The expression pattern of 55L and 97L in GIV-infected GK cells was first analysed by RT-PCR and then by Western blotting with the respective mAbs at 1, 3, 6, 12 and 24 hpi (Fig. 3). By RT-PCR analysis, the transcript of 97L was detected at as early as 3 hpi, whereas 55L at 6 hpi (Fig. 3a). At the protein level, 97L was detected at 6 h after infection, and 55L was not detected until 12 hpi (Fig. 3b). The result demonstrates that the two mAbs can be used as an effective detection tool in Western blotting. Based on the RT-PCR and Western blotting analyses, GIV 97L might be an Ó 2014 John Wiley & Sons Ltd
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immediate early gene and 55L a late one. To further verify the nature of these two viral proteins, the expression of 55L and 97L was assayed in GIV-infected cells in the presence of CHX or AraC. CHX is an inhibitor of de novo protein synthesis, and AraC is a pyrimidine antimetabolite that can block DNA synthesis. As shown in Fig 3c,d, the transcription and protein synthesis of 97L were not inhibited by CHX or Arac, but 55L and 45R (MCP – major capsid protein, a late gene) proteins were inhibited by CHX and AraC. The data support the notion that GIV 97L is an immediate early gene and 55L a late one. Intracellular localization of 55L and 97L proteins We also verified whether the 55L and 97L mAbs could be used to detect the intracellular localization of 55L and 97L by immunofluorescence microscopy. As shown in Fig. 4, the presence of 97L in the GIV-infected GK cells was recognized by GIV97L-mAb-3 at as early as 6 hpi, whereas the presence of 55L was not detected by GIV55L-mAb-2 until 12 hpi. Interestingly, the green fluorescence signals of GIV 55L formed aggregates mainly in cytoplasm, but the GIV 97L signals distributed at both the nucleus and cytoplasm of GIV-infected GK cells (Fig. 4). Overall, these results clearly demonstrated that GIV55L-mAb-2 and GIV97L-mAb-3 were able to discriminate GIV-infected cells from uninfected cells, suggesting that the two mAbs can be developed into a diagnostic tool for GIV infection. Discussion
Monoclonal antibodies (mAbs) are a powerful tool for the detection and characterization of virus (Pal et al. 2013; Zhang et al. 2013a,b). In recent years, mAb technology has had an important impact on aquaculture disease diagnosis (Lai et al. 2001a, 2002; Shi et al. 2003; C^ote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In this study, we have developed and characterized several mouse mAbs that are specific to the 55L or 97L proteins of GIV. Six mAbs were successfully developed against 55L and 8 mAbs against 97L, and the specificity of these mAbs was subsequently confirmed by ELISA and Western
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Journal of Fish Diseases 2015, 38, 249–258
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Figure 3 Temporal expression of GIV 55L and 97L transcripts and proteins in GIV-infected cells. GK cells were infected with GIV at an MOI of 5, and the transcripts (a) and proteins (b) were measured by RT-PCR and Western blotting, respectively, at 1, 3, 6, 12 and 24 hpi. The temporal expression of 55L and 97L was further analysed in GIV-infected cells at the presence of CHX or AraC. (a) Temporal expression of GIV mRNA: 97L and 55L were detected at as early as 3 and 6 hpi, respectively. (b) Temporal expression of GIV proteins: 97L and 55L were detected at 6 and 12 hpi, respectively. (c) Effect of CHX on the expression of 97L and 55L: the expression of 55L but not 97L was inhibited by CHX. GIV 45R (MCP) was included in the assay for comparison. (d) Effect of AraC on the expression of 97L and 55L: the expression of 55L and 45R (MCP) but not 97L was inhibited by AraC.
blotting assays. Among the mAbs, GIV55L-mAb2 and GIV97L-mAb-3 were of the highest affinity (OD650 nm >2) and were further characterized as IgG1 isotype. Further analyses demonstrated that these two mAbs could serve as potent tools to investigate the expression and potential role of 55L and 97L during GIV infection. For viruses such as GIV, transcription of viral genes can be classified into immediate early (IE), early (E) and late (L) genes according to their temporal synthesis (Williams 1996; Teng et al. 2008). By definition, the expression of IE genes relies solely on host proteins. The IE proteins are often proteins essential for the viral life cycle (Willis & Granoff 1985; Xia et al. 2010), whose function might be involved in activating the expression of viral early and late genes, altering the functions of host genes and eliminating host immune defence (Buisson et al. 1989; HolleyGuthrie et al. 1990; Williams et al. 2005; Chen et al. 2006; Huang et al. 2011). On the other hand, late genes encode proteins that usually are the viral structural proteins (Wan et al. 2010). Expression of late proteins often occurs after the replication of viral genome. Iridoviral MCP (major capsid protein) is an example of late gene, whose sequence has been commonly used to analyse the phylogenetic relationships between Ó 2014 John Wiley & Sons Ltd
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iridoviruses (Do et al. 2005a,b; L€ u et al. 2005; Go et al. 2006 and Imajoh, Ikawa & Oshima 2007). Being the most abundant component of a virion, MCP is often chosen as a target for the detection of iridoviral infection (Huang et al. 2004; Chinchar et al. 2005, 2009). However, MCP is a late gene, meaning that it cannot be used as an indicator of the early stage of a viral infection event. In this study, we have established two highly specific monoclonal antibodies against GIV 55L and 97L. The temporal expression of 55L and 97L as analysed by RT-PCR showed that 97L was detected at as early as 3 hpi, whereas 55L was not observed until 6 hpi. The data indicate that 97L might be an IE or E gene and 55L a late one. With the availability of the two newly established monoclonal antibodies, we were able to verify the temporal expression profile of 55L and 97L. We found that GIV 97L protein was detected by Western blotting at 6 hpi, but both 55L and 45R (MCP) were not detected until 12 hpi. Furthermore, only 97L but not 55L or 45R (MCP) was detected when viral genome replication was inhibited by the addition of AraC. These data consistently support the notion that GIV 97L is an IE gene and GIV 55L is an L gene during GIV replication. The data also
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demonstrated the validity of the two monoclonal antibodies as a useful analytical tool for GIV infection, especially the 97L monoclonal antibody being a marker of the early stage of GIV infection. In addition to the above application, we have demonstrated the feasibility of the two monoclonal antibodies in the application of immunofluorescence assay. As illustrated in Fig. 4, the subcellular localization of GIV 97L and 55L was found to be in the cytoplasm and nucleus, and the cytoplasm, respectively. The nuclear localization of 97L indicates that 97L is an IE protein whose function might be involved in the regulation of host or viral gene expression. This aspect of 97L requires further investigation in the future. In conclusion, we have generated two mouse mAbs specific to GIV 55L and 97L, respectively. The mAbs have been shown to be an effective diagnostic tool for GIV infection. In addition, the Ó 2014 John Wiley & Sons Ltd
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Figure 4 Intracellular localization of GIV 55L and 97L in infected GK cells. GK cells were infected with GIV at an MOI of 5, and the cells were fixed at 12 hpi, stained with specific mAbs. (a) The localization of GIV 55L in GIV-infected cells. (b) The localization of GIV 97L in GIV-infected cells. The nucleus was counter-stained by DAPI. The fluorescent signal was examined under a fluorescence microscope (Axio Observer, Zeiss).
two mAbs could be valuable tools for the future study of the biological function and pathological significance of GIV 55L and 97L during viral infection. The introduction of the two mAbs will expand the research capacity on the pathogenicity of GIV. Acknowledgements This study was supported by Grant No. NSC-101-2815-C-197-011-B from the National Science Council, Taiwan. Publication History Received: 17 September 2013 Revision received: 15 December 2013 Accepted: 16 December 2013
This paper was edited and accepted under the Editorship of Professor Ron Roberts.
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Journal of Fish Diseases 2015, 38, 249–258
Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins S-L Hu1, C-J Liou2, Y-H Cheng1, J-C Yiu3, P P Chiou4 and Y-S Lai1 1 2 3 4
Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan Department of Horticulture, National Ilan University, Yilan, Taiwan Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Yilan, Taiwan
Abstract
Grouper iridovirus (GIV) is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. The study of GIV pathogenicity has been hampered by the lack of proper immunological reagents to study the expression and function of viral proteins in the infected cells. In this study, two mouse monoclonal antibodies (mAbs) against GIV 55L and 97L proteins were produced. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen these hybridomas, resulting in the identification of two high-affinity mAbs named GIV55L-mAb-2 and GIV97L-mAb-3, respectively. Both mAbs belong to the IgG1 isotype and were effective in detecting their respective target viral protein. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analyses of GIV-infected GK cells revealed that GIV 97L is an immediate early gene, whereas GIV 55L a late one. The localization of 55L and 97L in GIV-infected cells was further characterized by immunofluorescence microscopy with the mAbs. The 55L protein mainly aggregated in the cytoplasm while 97L distributed in both the nucleus and cytoplasm of the infected cells. These studies demonstrate the validity of the two mAbs as immunodiagnostic and research reagents. Correspondence Y-S Lai, Department of Biotechnology and Animal Science, National Ilan University, 1, Sec. 1, Shen-Lung Road, Yilan 26047, Taiwan (e-mail: [email protected]) and P P Chiou, Marine Research Station, Institute of Cellular & Organismic Biology, Academia Sinica, 23-10 Dawun Road, Jiaoxi, Yilan 26242, Taiwan (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Keywords: ELISA, grouper iridovirus, immunofluorescence, monoclonal antibodies.
Introduction
Grouper (Epinephelus spp) is one of the most economically important cultured marine fish in many Asian countries. A major constraint in grouper hatchery production and farming is infectious disease (Chi 1997; Lai et al. 2001a,b). Iridovirus is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages (Lai et al. 2000, 2003). Iridoviruses are large, icosahedral, double-stranded DNA virus with a viral particle size ranging from 120 to 350 nm in diameter (Williams 1996; Chinchar et al. 2005). The iridoviridae family comprises five genera: iridovirus, chloriridovirus, ranavirus, lymphocystivirus and megalocytivirus, whose members can infect invertebrates and vertebrates (Williams et al. 2000; Williams, Barbosa-Solomieu & Chinchar 2005; Eaton et al. 2007). Vertebrate iridoviruses have been reported in fish, amphibians and reptiles, and many of them can lead to severe systemic diseases in their hosts (Williams 1996). Grouper iridovirus (GIV), originally isolated from southern Taiwan, is a member of the ranavirus genus and has resulted in significant economic losses in the grouper aquaculture industry (Lai et al. 2000; Murali et al. 2002). The genome of GIV consists of 139 793 bp in length with a 49% G + C content. The genome is predicted to encode 120 open reading frames (ORFs), whose sizes ranging from 62 to 1268 amino acids (Tsai
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et al. 2005). Despite the devastating impact of iridoviruses on the grouper aquaculture industry, the study on the mechanism of iridoviral pathogenicity remains difficult due to the lack of proper tools. In an earlier investigation, we established 7 GIV-susceptible cell lines from yellow grouper, Epinephelus awoara (Lai et al. 2003) and two cell lines from Barramundi, Lates calcarifer, in which GIV was capable of inducing apoptosis (Lai et al. 2008). These cell lines have been shown to be valuable tools to assist in the investigation on the cell death caused by GIV (Chiou, Chen & Lai 2009; Pham et al. 2012). Yet, other tools such as immunological reagents are still in need to assist in the study of the pathogenicity mechanism of iridoviruses. Monoclonal antibodies (mAbs) are efficient tools for detection, screening and characterization of biomolecules and have wide applications in disease diagnosis and immunotherapy (Shepard et al. 1991; Toi et al. 2004). In virus research, specific mAbs are widely used in diagnosis, virus pathogenesis and immunotherapy of viral disease (Shi et al. 2003; Fofana et al. 2013; Wang et al. 2013; Zhang et al. 2013a,b). The aim of this study was to produce mAbs that could assist in the immunological detection of GIV infection. We showed here the development and characterization of two mAbs against GIV 55L and 97L. These two mAbs were specific to their target antigens, and their ability to serve as detection tools for GIV infection was demonstrated by enzymelinked immunosorbent assay (ELISA), immunoblotting and immunofluorescence analyses.
Materials and methods
Virus and cells GK (grouper kidney) cells (Lai et al. 2000) were grown at 28 °C in Leibovitz’s L15 medium supplemented with 10% foetal bovine serum (FBS), 100 IU mL 1 penicillin and 100 lg mL 1 streptomycin. The propagation and purification of GIV was conducted in GK cells as described previously (Lai et al. 2000). Construction of expression plasmids The complete ORFs of GIV 55L and GIV 97L were amplified by PCR from GIV genomic DNA, using oligonucleotide primers containing designed restriction enzyme cleavage sites. The following Ó 2014 John Wiley & Sons Ltd
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primers were used for GIV 55L: forward5′-CGCGGATCCATGGATCAGTGGCTG-3′ (BamH Ι site underlined), and reverse-5′-CCG CTCGAGTTTAGTCTCCATCAA-3′ (Xho Ι site underlined). PCR was carried out under the following conditions: 10 min at 95 °C for 1 cycle; 1 min at 95 °C, 1 min at 56 °C and 1 min at 72 °C, for 35 cycles; 10 min at 72 °C for 1 cycle. The following primers were used for GIV 097L: forward-5′-CGCGGATCCATGGAATGTTTATA C-3′ (BamH Ι site underlined), reverse-5′-CCGC TCGAGCACAAACCCAAATTT-3′ (Xho Ι site underlined). PCR was carried out under the following conditions: 5 min at 95 °C for 1 cycle; 30 s at 95 °C, 40 s at 55 °C and 1 min at 72 °C, for 35 cycles; 10 min at 72 °C for 1 cycle. The amplified fragment of GIV 55L was cloned into the pGEX-6P-1 expression vector and GIV 97L into the pET23a expression vector. These constructs were transformed into E.coli XL1-Blue, and the transformation was confirmed by restriction enzyme digestion and DNA sequencing. Expression and purification of recombinant proteins After transformation into BL21 (DE3) cells, a single positive clone was inoculated in 3 mL of Luria– Bertani (LB) broth containing 100 lg mL 1 ampicillin and cultured overnight at 37 °C. The overnight culture of E. coli cells was inoculated into 500 mL LB supplemented with 100 lg mL 1 of ampicillin. When the OD600 reached 0.5, IPTG was added at a final concentration of 1 mM to induce protein expression for 24 h. The cells (pET23a-97L) were harvested by centrifugation (4000 g, 20 min), and the pellet was resuspended in lysis buffer A (6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0; 5 mL buffer A/1 g cell pellet) overnight at 4 °C. The cells were disrupted by lysis buffer A, and the insoluble debris was removed by centrifugation at 10 000 g for 30 min. The supernatant was then applied directly onto a Ni-NTA agarose affinity chromatography column and washed sequentially by lysis buffer A containing 1, 5, 10, 20 and 200 mM imidazole. The elute was collected in 3-mL fractions and analysed by SDS-PAGE. The fractions containing protein of the expected molecular weight were pooled and dialysed with S100 buffer (25 mM HEPES, 20% Glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM DTT, pH 7.9). The
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pGEX-6p-1-55L recombinant proteins were purified using GST-agarose (Fluka) following the manufacturer’s protocol. After dialysis, the concentration of purified recombinant protein was measured using a Bradford assay. Hybridoma preparation BALB/c mice (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used for immunization. Each mouse (8 weeks old) was injected intraperitoneally with 100 lg of recombinant protein, which was emulsified with an equal volume of Freund’s complete adjuvant. After 14 days, a booster injection of the same dosage, but prepared in Freund’s incomplete adjuvant, was given as described above. Two more injections were administered at 2-week intervals. Two days after the last injection, spleen cells were removed and fused with mouse myeloma cells (NS1) in the presence of 50% polyethylene glycol (MW 1500). The fused cells were then resuspended in RPMI1640 medium supplemented with 10% FBS, 19 hypoxanthine–aminopterin–thymidine (HAT media supplement (509) Hybri-MaxTM, Sigma). Seven to ten days after fusion, the culture medium from hybridoma cells was screened for the presence of specific antibodies by ELISA. Cells in the positive wells were further subcloned by limiting dilution and screened by enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assay ELISA plates (Nunc) were coated with 100 lL of antigen protein suspension (1 ng lL 1) per well diluted in PBS containing 0.05% NaN3 and were incubated at 4 °C overnight. The plates were then blocked with 5% skim milk in PBS for 2 h at 37 °C and washed three times with PBST [PBS containing 0.05% (v/v) Tween-20]. One hundred microlitres of mouse serum and hybridoma supernatant diluted in PBST was added to each well. After 2-h incubation at 37 °C, plates were rinsed three times with PBST, and 100 lL of 1:5000-diluted horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (Santa Cruz Biotechnology) was added to each well. The plates were incubated for 1 h at 37 °C. After washing the plate three times with PBST, the enzyme activity was determined by adding TMB single solution substrate (Invitrogen). After 30-min Ó 2014 John Wiley & Sons Ltd
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incubation in dark, the light absorbance of each well was read at 650 nm with an ELISA plate reader (SpectraMax M2, Molecular Devices). Mouse mAb Isotype determination and purification The isotyping of the established mAbs was conducted by ELISA with a mouse mAb isotyping kit (Sigma). Each BALB/c mice (20 weeks old) was injected intraperitoneally with 200-lL pristine, and 10 days later, 5 9 106 hybridoma cells would be injected into the mouse. The ascitic fluids were subsequently collected for mAbs purification using Protein G-agarose (Thermo, NAbTM protein G spin kit) following the manufacturer’s protocol. The animal experiments and ascites production were conducted by following the Institutional Animal Care and Use Committee (IACUC) guidelines of the National Laboratory Animal Center (Taiwan). Reverse-transcription polymerase chain reaction (RT-PCR) GK cells were infected with GIV at an MOI of 5 and harvested at 0, 1, 3, 6, 12 and 24 h postinfection (hpi) in the presence or absence of cycloheximide (CHX; 50 g mL 1; Sigma Aldrich, St. Louis, MO). The total RNA was extracted using TRIzol reagent (Invitrogen) with RNase-free DNase (New England Biolabs) to remove contaminated DNA. Two micrograms of the total RNA was subsequently reverse-transcribed into the firststranded cDNA using reverse transcriptase (Roche) with random primers, following the manufacturer’s protocol. PCR was carried out under the following conditions: 2 lL of cDNA, 2 lM forward primer, 2 lM reverse primer, 2.5 lM of each dNTP, 19 PCR buffer and 2.5 U Tag DNA polymerase (Viogene) to a final volume of 50 lL. After amplification, the PCR products were electrophoresed in a 1.2% agarose-TAE buffer gel and stained with ethidium bromide. Western blotting Western blotting was performed with lysates collected from GIV-infected GK cells at an MOI of 5 in the presence or absence of cytosine arabinoside (AraC; 40 lg mL 1; Sigma Aldrich). At 1 h, 3 h, 6 h, 12 h and 24 hpi, GIV-infected or
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mock-infected cells were rinsed with PBS and lysed in 29 SDS/sample buffer (100 mM Tris– HCl (pH 6.8), 10% b-mercaptoethanol, 4% SDS, 0.2% bromophenol blue and 20% glycerol). Cell lysates were subjected to 12% SDS-PAGE (120 V, 1.5 h) and transferred onto PVDF membrane (Pall Corporation) at 400 mA for 2 h in a Bio-Rad mini Trans-Blot electrophoretic transfer cell for Western blotting analysis. The membrane was washed in PBST and blocked with 5% skimmed milk overnight at 4 °C. After washing, the membrane was reacted with GIV55L-mAb-2 (500 lg mL 1, 1:5000), GIV97L-mAb-3 (500 lg mL 1, 1:5000), GIV45R (MCP) (mouse serum, 1:5000) or b-actin (3.1 mg mL 1, 1:5000) (Sigma, catalog A5441) for 2 h at 37 °C, washed again and then incubated with alkaline phosphatase-conjugated goat anti-mouse immunoglobulins (400 lg mL 1, 1:5000) (catalog 2058; Santa Cruz Biotechnology) at 37 °C for 1 h. Specific bands were visualized with 5-bromo-4chloro-3-indolyl phosphate (BCIP) and 4-nitroblue tetrazolium chloride (NBT). Immunofluorescent microscopy GK cells grown on Lab-Tek chamber slideTM (four-well Permanox Slide, Nalge Nunc International Corp.) were infected with GIV at an MOI of 5. At 1, 6 and 12 hpi, the culture media were removed and cells were washed with PBS. The cells were then fixed in 2% para-formaldehyde/ 0.1% Triton X-100 for 30 min on ice. After removal of fixative, the cells were washed twice in ice-cold PBS and incubated with designated specific mAb (500 lg mL 1, 1:100) (GIV55L-mAb2 or GIV97L-mAb-3) at 4 °C for 60 min. After wash with ice-cold PBS three times, the fluorescein-labelled goat anti-mouse IgG (H + L) (500 lg mL 1, 1:50) (KPL catalog 02-18-06) was added into the chamber slide and incubated at 4 °C for 60 min. The slides were washed with ice-cold PBS three times, mounted and subjected to examination under a fluorescence microscope (Axio Observer, Zeiss).
Results
Sequence analysis of GIV 55L and 97L GIV 55L (GenBank accession no. AAV91073) is 1128 bp in length, encoding a putative protein of Ó 2014 John Wiley & Sons Ltd
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376 amino acids with a predicted molecular mass of 41.57 kDa. The deduced amino acid sequence of GIV 55L is 99% identical to the homologs of Singapore grouper iridovirus (SGIV) (AAS18099) and 47% to frog virus 3 (FV3) (AAT09740), Ambystoma tigrinum virus (ATV) (AAP33202) and tiger frog virus (TFV) (AAL77809) (Fig. 1a). The predicated protein structure of GIV55L and the homologs of other iridoviruses suggest that it is an RNAse III (Fig. 1). GIV 97L (GenBank accession no. AAV91107) is of 975 bp and encodes a putative protein of 325 amino acids with a predicted molecular mass of 36.68 kDa. The deduced amino acid sequence of GIV 97L is 98, 45, 44 and 44% identical to the homologs of SGIV (AAS18161), FV3 (AAT09733), ATV (AA P33208) and TFV (AAL77808), respectively (Fig. 1b). Expression and purification of recombinant GIV 55L and 97L proteins Plasmids pGEX-6P-1-55L and pET23a-97L were constructed to express recombinant GST-55L and His-97L proteins, respectively. The estimated size of recombinant GST-55L fusion protein was 67.57 kDa, and that of His-97L, 36.68 kDa. As shown in Fig. 2, at 24 h of induction with 1 mM IPTG, the two recombinant proteins were successfully expressed, as demonstrated by the presence of bands of the respective estimated size. Production and characterization of GIV 55L and 97L mAbs To produce GIV 55L and 97L monoclonal antibodies, NS1 myeloma cells were fused with spleen cells removed from four mice immunized with purified recombinant GST-55L and His-97L proteins, respectively. The fused cells were subsequently screened by ELISA against recombinant 55L and 97L. Six positive clones (GIV55L-mAb1, GIV55L-mAb-2, GIV55L-mAb-3, GIV55LmAb-4, GIV55L-mAb-5 and GIV55L-mAb-6) were identified against GIV 55 of 52 clones, and 8 positive clones (GIV97L-mAb-1, GIV97L-mAb2, GIV97L-mAb-3, GIV97L-mAb-4, GIV97LmAb-5, GIV97L-mAb-6, GIV97L-mAb-7 and GIV97L-mAb-8) against GIV 97L identified of 85. During the screening process, the strategy of limiting dilution was adopted and repeated three times. By ELISA analysis, clones GIV55L-mAb-2
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(a)
(b)
Figure 1 Multiple amino acid sequence alignment of 55L and 97L of GIV with related genes sequences of four other iridoviruses. The highly conserved residues in all sequences are marked by a pound symbol under the alignment. (a) GIV 55L amino acid sequence alignment. (b) GIV 97L amino acid sequence alignment.
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(a)
1
2
3
(b)
4
KDa
43 ─ 34 ─ 26 ─
2
3
4
KDa
95 ─ 72 ─ 55 ─
1
GST-55L
95 ─ 72 ─ 55 ─ 43 ─
His-97L
34 ─ 26 ─
17 ─
Figure 2 Purification of recombinant GIV 55L and 97L proteins. Recombinant proteins were harvested from transformed E. coli, and the proteins were further purified by GST or Ni-NTA Agarose affinity chromatography column. (a) Recombinant GST-55L: lane 1, prestained protein marker; lane 2, E. coli BL21(DE3)-pGEX-6p-1-55L; lane 3, E coli BL21 (DE3)-pGEX-6p-1-55L/IPTG; lane 4, purified recombinant GST-55L. (b) Recombinant 97L: lane 1, prestained protein marker; lane 2, E. coli BL21(DE3)-pET23a-97L; lane 3, E. coli BL21(DE3)-pET23a-97L/IPTG; lane 4, purified recombinant His-97L.
and GIV97L-mAb-3 showed the greatest reactivity against 55L and 97L, respectively. Thus, the GIV55L-mAb-2 and GIV97L-mAb-3 hybridoma cells were injected into mice, from which 55L- and 97L-specific mAbs were successfully produced (data not showed). Further isotyping showed that both 55L and 97L mAbs were of IgG1 isotype (data not showed). Expression pattern of 55L and 97L over the course of the GIV infection Experiments were carried out to verify the feasibility of the two newly established mAbs as immunological reagents for detecting 55L and 97L in GIV-infected cells. The expression pattern of 55L and 97L in GIV-infected GK cells was first analysed by RT-PCR and then by Western blotting with the respective mAbs at 1, 3, 6, 12 and 24 hpi (Fig. 3). By RT-PCR analysis, the transcript of 97L was detected at as early as 3 hpi, whereas 55L at 6 hpi (Fig. 3a). At the protein level, 97L was detected at 6 h after infection, and 55L was not detected until 12 hpi (Fig. 3b). The result demonstrates that the two mAbs can be used as an effective detection tool in Western blotting. Based on the RT-PCR and Western blotting analyses, GIV 97L might be an Ó 2014 John Wiley & Sons Ltd
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immediate early gene and 55L a late one. To further verify the nature of these two viral proteins, the expression of 55L and 97L was assayed in GIV-infected cells in the presence of CHX or AraC. CHX is an inhibitor of de novo protein synthesis, and AraC is a pyrimidine antimetabolite that can block DNA synthesis. As shown in Fig 3c,d, the transcription and protein synthesis of 97L were not inhibited by CHX or Arac, but 55L and 45R (MCP – major capsid protein, a late gene) proteins were inhibited by CHX and AraC. The data support the notion that GIV 97L is an immediate early gene and 55L a late one. Intracellular localization of 55L and 97L proteins We also verified whether the 55L and 97L mAbs could be used to detect the intracellular localization of 55L and 97L by immunofluorescence microscopy. As shown in Fig. 4, the presence of 97L in the GIV-infected GK cells was recognized by GIV97L-mAb-3 at as early as 6 hpi, whereas the presence of 55L was not detected by GIV55L-mAb-2 until 12 hpi. Interestingly, the green fluorescence signals of GIV 55L formed aggregates mainly in cytoplasm, but the GIV 97L signals distributed at both the nucleus and cytoplasm of GIV-infected GK cells (Fig. 4). Overall, these results clearly demonstrated that GIV55L-mAb-2 and GIV97L-mAb-3 were able to discriminate GIV-infected cells from uninfected cells, suggesting that the two mAbs can be developed into a diagnostic tool for GIV infection. Discussion
Monoclonal antibodies (mAbs) are a powerful tool for the detection and characterization of virus (Pal et al. 2013; Zhang et al. 2013a,b). In recent years, mAb technology has had an important impact on aquaculture disease diagnosis (Lai et al. 2001a, 2002; Shi et al. 2003; C^ote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In this study, we have developed and characterized several mouse mAbs that are specific to the 55L or 97L proteins of GIV. Six mAbs were successfully developed against 55L and 8 mAbs against 97L, and the specificity of these mAbs was subsequently confirmed by ELISA and Western
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Figure 3 Temporal expression of GIV 55L and 97L transcripts and proteins in GIV-infected cells. GK cells were infected with GIV at an MOI of 5, and the transcripts (a) and proteins (b) were measured by RT-PCR and Western blotting, respectively, at 1, 3, 6, 12 and 24 hpi. The temporal expression of 55L and 97L was further analysed in GIV-infected cells at the presence of CHX or AraC. (a) Temporal expression of GIV mRNA: 97L and 55L were detected at as early as 3 and 6 hpi, respectively. (b) Temporal expression of GIV proteins: 97L and 55L were detected at 6 and 12 hpi, respectively. (c) Effect of CHX on the expression of 97L and 55L: the expression of 55L but not 97L was inhibited by CHX. GIV 45R (MCP) was included in the assay for comparison. (d) Effect of AraC on the expression of 97L and 55L: the expression of 55L and 45R (MCP) but not 97L was inhibited by AraC.
blotting assays. Among the mAbs, GIV55L-mAb2 and GIV97L-mAb-3 were of the highest affinity (OD650 nm >2) and were further characterized as IgG1 isotype. Further analyses demonstrated that these two mAbs could serve as potent tools to investigate the expression and potential role of 55L and 97L during GIV infection. For viruses such as GIV, transcription of viral genes can be classified into immediate early (IE), early (E) and late (L) genes according to their temporal synthesis (Williams 1996; Teng et al. 2008). By definition, the expression of IE genes relies solely on host proteins. The IE proteins are often proteins essential for the viral life cycle (Willis & Granoff 1985; Xia et al. 2010), whose function might be involved in activating the expression of viral early and late genes, altering the functions of host genes and eliminating host immune defence (Buisson et al. 1989; HolleyGuthrie et al. 1990; Williams et al. 2005; Chen et al. 2006; Huang et al. 2011). On the other hand, late genes encode proteins that usually are the viral structural proteins (Wan et al. 2010). Expression of late proteins often occurs after the replication of viral genome. Iridoviral MCP (major capsid protein) is an example of late gene, whose sequence has been commonly used to analyse the phylogenetic relationships between Ó 2014 John Wiley & Sons Ltd
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iridoviruses (Do et al. 2005a,b; L€ u et al. 2005; Go et al. 2006 and Imajoh, Ikawa & Oshima 2007). Being the most abundant component of a virion, MCP is often chosen as a target for the detection of iridoviral infection (Huang et al. 2004; Chinchar et al. 2005, 2009). However, MCP is a late gene, meaning that it cannot be used as an indicator of the early stage of a viral infection event. In this study, we have established two highly specific monoclonal antibodies against GIV 55L and 97L. The temporal expression of 55L and 97L as analysed by RT-PCR showed that 97L was detected at as early as 3 hpi, whereas 55L was not observed until 6 hpi. The data indicate that 97L might be an IE or E gene and 55L a late one. With the availability of the two newly established monoclonal antibodies, we were able to verify the temporal expression profile of 55L and 97L. We found that GIV 97L protein was detected by Western blotting at 6 hpi, but both 55L and 45R (MCP) were not detected until 12 hpi. Furthermore, only 97L but not 55L or 45R (MCP) was detected when viral genome replication was inhibited by the addition of AraC. These data consistently support the notion that GIV 97L is an IE gene and GIV 55L is an L gene during GIV replication. The data also
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demonstrated the validity of the two monoclonal antibodies as a useful analytical tool for GIV infection, especially the 97L monoclonal antibody being a marker of the early stage of GIV infection. In addition to the above application, we have demonstrated the feasibility of the two monoclonal antibodies in the application of immunofluorescence assay. As illustrated in Fig. 4, the subcellular localization of GIV 97L and 55L was found to be in the cytoplasm and nucleus, and the cytoplasm, respectively. The nuclear localization of 97L indicates that 97L is an IE protein whose function might be involved in the regulation of host or viral gene expression. This aspect of 97L requires further investigation in the future. In conclusion, we have generated two mouse mAbs specific to GIV 55L and 97L, respectively. The mAbs have been shown to be an effective diagnostic tool for GIV infection. In addition, the Ó 2014 John Wiley & Sons Ltd
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Figure 4 Intracellular localization of GIV 55L and 97L in infected GK cells. GK cells were infected with GIV at an MOI of 5, and the cells were fixed at 12 hpi, stained with specific mAbs. (a) The localization of GIV 55L in GIV-infected cells. (b) The localization of GIV 97L in GIV-infected cells. The nucleus was counter-stained by DAPI. The fluorescent signal was examined under a fluorescence microscope (Axio Observer, Zeiss).
two mAbs could be valuable tools for the future study of the biological function and pathological significance of GIV 55L and 97L during viral infection. The introduction of the two mAbs will expand the research capacity on the pathogenicity of GIV. Acknowledgements This study was supported by Grant No. NSC-101-2815-C-197-011-B from the National Science Council, Taiwan. Publication History Received: 17 September 2013 Revision received: 15 December 2013 Accepted: 16 December 2013
This paper was edited and accepted under the Editorship of Professor Ron Roberts.
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