doi:10.1111/jfd.12331
Journal of Fish Diseases 2016, 39, 129–141
Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L Z-Y Chen1, P P Chiou1,2, C-J Liou3,4 and Y-S Lai1 1 2 3 4
Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan
Abstract
Viral envelope proteins play important roles in viral infection and assembly. The grouper iridovirus ORF 64L (GIV-64L) was predicted to encode an envelope protein and was conserved in all sequenced Ranaviruses. In this study, the complete nucleotide sequence of the GIV-64L gene (1215 bp) was cloned into the isopropyl b-D-1thiogalactopyranoside (IPTG) induction prokaryotic expression vector pET23a. The approximately 50.2 kDa recombinant GIV-64L-His protein was induced, purified and used as an immunogen to immunize BALB/c mice. Three monoclonal antibodies (mAbs), all IgG1 class antibodies against GIV-64L protein, were produced by enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction analyses revealed GIV-64L to be a late gene when expressed in grouper kidney cells during GIV infection with cycloheximide (an inhibitor of protein synthesis) or cytosine arabinoside (an inhibitor of DNA synthesis) present. Finally, one of the established mAbs, GIV-64L-mAb-17, was used in Western blotting and an immunofluorescence assay, which showed that GIV-64L protein was expressed at 24 h post-infection and localized only in the cytoplasm in GIV-infected cells, packed into a whole virus particle. The presently characterized GIV-64L mAbs should have 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]) Ó 2015 John Wiley & Sons Ltd
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widespread applications in GIV immunodiagnostics and other research, and these results should offer important insights into the pathogenesis of GIV. Keywords: GIV-64L, Grouper iridovirus, immunofluorescence, monoclonal antibody. Introduction
Grouper (Epinephelus spp.) is one of the most economically important cultured marine fish in many Asian countries. Viral disease is a major constraint in the hatchery production of grouper, and iridoviruses are among the most important viral pathogens in these fish, particularly at the fry and fingerling stages. Iridoviruses are large icosahedral viruses containing a linear double-stranded DNA packed in a viral particle with diameters ranging from 120 to 350 nm (Williams 1996). The Iridoviridae family is classified into five genera— Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus and Megalocytivirus—whose members can infect only ectothermic vertebrates and invertebrates (Chinchar et al. 2005; Williams, BarbosaSolomieu & Chinchar 2005). Grouper infected with iridovirus might show darkened body colour, pale gills and an enlarged spleen, and often become lethargic with low appetite (Langdon & Humphrey 1987; Schuh & Shirley 1990; Chua et al. 1994; He et al. 2000; Lai et al. 2000). Microscopic pathological signs might include enlargement of cells and necrosis of renal and splenic hematopoietic tissues (Qin et al. 2003).
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Grouper iridovirus (GIV), a member of the Ranavirus genus that was originally isolated from southern Taiwan, has caused significant economic losses in the grouper aquaculture industry for years (Murali et al. 2002; Tsai et al. 2005). GIVsusceptible cell lines from yellow grouper have been established (Lai et al. 2000, 2003), and the complete genome of GIV has been sequenced and consists of 139 793 bp with a 49% G + C content. The genome is predicted to encode 120 open reading frames (ORFs), ranging in size from 62 to 1268 amino acids (Tsai et al. 2005). In recent years, although the GIV genome sequence, host cellular gene expression (Wu et al. 2012) and apoptosis characteristics have been investigated (Lai et al. 2008; Chiou, Chen & Lai 2009; Pham et al. 2012), the understanding of the molecular aspects of GIV pathogenicity is still limited. Four GIV genes have been identified and characterized: GIV-45R (major capsid protein) (Lin et al. 2014), GIV-49L (predicted function and/or similarity: purine nucleoside phosphorylase) (Ting et al. 2004), GIV-55L (predicted function and/or similarity: RNase III) and GIV-97L (predicted function and/or similarity: NTPase–helicase) (Hu et al. 2014). Mouse monoclonal antibodies (mAbs) are efficient tools for detecting and characterizing viral pathogens. In recent years, mAb technology has had an important effect on aquaculture disease diagnosis (Lai et al. 2001, 2002; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). To understand the molecular mechanism of GIV pathogenesis, the expression pattern and assay tools of viral genes must be developed and evaluated. Viral envelope proteins are critical to virus infection and virus–host interaction, and their specific antibodies can serve as a valuable tool for virus detection. The GIV-64L was predicated in silico to encode an envelope protein, thus could be a good candidate target of immunological detection of the presence of the virus. In this study, we cloned the GIV-64L gene from GIV genomic DNA and produced mouse mAbs specific to GIV-64L. This investigation marks the first examination of GIV-64L gene expression and subcellular protein location during GIV infection in vitro, and these specific mAbs are a valuable tool for GIV diagnostics and studies on GIV pathogenesis. Ó 2015 John Wiley & Sons Ltd
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Z-Y Chen et al. Mouse mAbs against GIV-64L
Materials and methods
Cells and viruses Grouper kidney (GK) cells previously established and characterized from the kidney mass of grouper were used in this study (Lai et al. 2000). The propagation and purification of GIV was conducted in GK cells as described previously (Lai et al. 2000). GK cells (107) growth in a 175-cm2 flask was infected with GIV at a multiplicity of infection (MOI) of 0.01. The virus was then allowed to multiply for 7 days at 28 °C. The culture mediums of 10 flasks were collected and centrifuged at 2000 9 g for 30 min at 4 °C. The supernatant and pellet were collected separately. The supernatant was mixed with polyethylene glycol (PEG, Sigma), stirred at 4 °C overnight and then centrifuged at 4000 9 g for 1 h at 4 °C. The pellet was resuspended in 2 mL of TNE buffer (50 mM Tris–HCl, 1 mM EDTA (ethylene diamine tetraacetic acid), 100 mM NaCl, pH 7.3), mixed with an equal volume of Freon (1,1,2-trichlorotrifluoroethane, Sigma) and centrifuged at 1800 9 g for 10 min. The pellet (cell-associated virus) was also extracted with Freon in the same way. Aqueous solutions of both virus samples were collected and combined, layered over a three-step caesium chloride gradient (4 mL 40%, 3 mL 30%, 2 mL 20%) and centrifuged at 13 000 9 g for 16 h at 4 °C. The virus band was collected and diluted with 5 mL TNE buffer and recentrifuged at 23 000 9 g for 2 h at 4 °C. The purified virus pellet was resuspended in 1 mL of TNE buffer and used for all analyses. The virus titre was calculated by the TCID50 method, described in Reed & Muench (1938). Computer-assisted analysis for protein alignment and phylogenetic tree generation GIV-64L homology analyses were performed using the BLAST network server of the National Center for Biotechnology Information. The multiple alignment of the GIV-64L amino acid sequence with those of another 10 sequenced iridoviruses was edited and performed using the GeneDoc program. The phylogenetic tree was generated using MEGA 6 software. The detail information for the other iridoviruses using in the blast analysis and phylogenetic tree was listed in Table 1.
Z-Y Chen et al. Mouse mAbs against GIV-64L
Journal of Fish Diseases 2016, 39, 129–141
pET23a-GIV-64L plasmid construction The full length of GIV-64L was isolated from GIV genomic DNA by polymerase chain reaction (PCR) using a pair of primers: GIV-64L-F/64L-R (Table 1). PCR was carried out under the following conditions: 10 min at 95 °C for one cycle; 1 min at 95 °C, 1 min at 56 °C and 1 min at 72 °C for 35 cycles; and 10 min at 72 °C for one cycle. The amplified fragment of GIV-64L was cloned into the pET23a (Novagen) expression vector using T4 DNA ligase (Roche). These constructs were transformed into Escherichia coli (E. coli) XL1-Blue, and the transformation was confirmed by restriction enzyme digestion and DNA sequencing. Preparation of recombinant GIV-64L-His protein After DNA sequencing, the recombinant fusion construct, pET23a-GIV-64L expression vector, was transformed into E. coli BL21(DE3)-competent cells, and recombinant GIV-64L-histidine tag (GIV-64L-His) protein was induced by 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) (Sigma) for 24 h at 37 °C. Thereafter, cells were cooled on ice for 30 min and harvested by centrifugation (4000 9 g, 20 min). 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 insoluble debris was removed by centrifugation at 10 000 9 g for 30 min, and the supernatant was collected and loaded onto a Ni-NTA resin affinity column (GEHealthcare). The column was successively washed with lysis buffer A containing 1, 10, 20 and 200 mM imidazole, respectively. The eluted fractions were
collected and analysed by 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie brilliant blue R-250. 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) until the imidazole was removed and stored at 20 °C until use. Immunization and antibody production Five female (8 weeks old) BALB/c mice (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used for immunization. For the first-time injection, each mouse was injected subcutaneously with 200 lL of emulsion containing 100 lg of GIV-64L-His and Freund’s complete adjuvant (Sigma). Then, the mouse was boosted with same volume of emulsion mixed 1:1 with incomplete Freund’s adjuvant (Sigma) at 2 weeks and again at 4 weeks. The mouse antiserum was collected every week to assess immunity by Western blotting and enzymelinked immunosorbent assay (ELISA). All the animal experiments were performed in accordance with the guidelines of the ethics review committee for animal experiments at National Ilan University. The protocols for mouse immunization and cell fusion were similar to those previously described (Hu et al. 2014). The selection of hybridoma clones producing mAbs specific against GIV-64L proteins was accomplished by ELISA and Western blotting. ELISA assay For ELISA, the ELISA 96-well plates (Nunc) were coated with 0.1 lg/well of recombinant GIV-64L-
Table 1 GenBank accession numbers of GIV-64L homologues used in this study
Ó 2015 John Wiley & Sons Ltd
Virus names
ORF
Accession no.
Identity (%)
Length a.a.
Grouper iridovirus (GIV) Singapore grouper iridovirus (SGIV) Tiger frog virus (TFV) Soft-shelled turtle iridovirus (SUV) Frog virus 3 (FV3) Common midwife toad ranavirus (CMTV) Andrias davidianus ranavirus (ADRV) Ambystoma tigrinum virus (ATV) European catfish virus (ECV) Chinese giant salamander iridovirus (CGSIV) Epizootic haematopoietic necrosis virus (EIINV)
64L 93L 23R 27R 23R 84L 87L 53R 75R 90L 55R
AAV91080 YP164188 ABB92288 ACF42246 YP031601 AFA44990 AGV20618 YP003826 YP006347667 AHA80935 ACQ25245
97 23 23 23 23 23 22 22 23 23
405 405 382 382 382 382 382 382 382 365 353
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3h
6h
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36 h
C
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3h
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GIV-64L GIV-45R (MCP) β-Actin
(b)
GIV-64L + CHX GIV-45R (MCP) + CHX GIV-64L + AraC GIV-45R (MCP) + AraC Figure 4 Temporal expression pattern of GIV-64L transcription compared with GIV-45R(MCP) was measured by RT-PCR during GIV infection of GK cells. (a) GK cells were infected with GIV at MOI = 5, and transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. (b) GK cells were infected with GIV at MOI = 5 with the presence of CHX or AraC, and the transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. Grouper iridovirus ORF 64L: (GIV64L); grouper iridovirus ORF 45R (major capsid protein): GIV-45R(MCP); cycloheximide: CHX; cytosine arabinoside: AraC.
(a)
kDa M
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GIV-64L 75 64 48
GIV-55L
35 GIV-45R(MCP) 25 17
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11 Figure 5 Temporal expression pattern of GIV-64L, GIV-45R(MCP) and GIV-55L proteins. (a) GK cells were infected with GIV at MOI = 5, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. Cell lysates were separated by SDS-PAGE. The gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker. (b) GK cells were infected with GIV at MOI = 5, and translation of GIV-64L, GIV-45R(MCP) and GIV-55L was measured by Western blotting analysis, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. GIV-64L protein was detected with GIV-64L-mAb-17. Grouper iridovirus ORF 55L: GIV-55L.
was consistent with the position of GIV in iridovirus evolution. Monoclonal antibodies are a good tool for detecting and characterizing viral infection (Patil et al. 2013; Zhang et al. 2013). In recent years, mAb technology has had an important impact on aquaculture disease diagnostics (Lai et al. 2001, Ó 2015 John Wiley & Sons Ltd
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2002; Shi et al. 2003; Cote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In our previous study, GIV-45R(MCP), GIV-55L and GIV-97L mAbs were produced and used successfully to verify the detection and subcellular localization of target viral proteins in GIV-infected GK cells. In
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immunoglobulins (400 lg mL1, 1:5000) (Santa Cruz) at 37 °C for 1 h. Finally, the colour band was developed in the dark using the substrates 4-nitro-blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate (NBT/BCIP) (Sigma). Immunofluorescence assay To analyse the location of the GIV-64L protein, GK cells were grown on a coverslip in a 12well plate and infected with GIV at a MOI = 5. At 0, 24 and 30 hpi, cells were fixed with 2% paraformaldehyde/0.1% Triton X-100 for 30 min at 37 °C. After removal of fixative, the coverslip was blocked for 30 min at 37 °C by bovine serum albumin. Cells were incubated with GIV-64L-mAb-17 (500 lg mL1, 1:100) for 1 h at 37 °C. After three washes with PBS, the fluorescein-labelled goat anti-mouse immunoglobulin G (H+L) (500 lg mL1, 1:50) (KPL) was added into the well and incubated for 1 h at 37 °C. Cellular and GIV DNA were labelled with 4, 6-diamidino-2-phenylindole (DAPI) for 10 min. The slides were washed with PBS three times, mounted and observed under a confocal fluorescence microscope (Olympus IX81, Japan).
Results
Identification and sequence analysis of GIV-64L GIV-64L (GenBank accession no. AAV91080) is 1215 bp in length, encoding a putative protein of 405 amino acids with a predicted molecular mass of 47.5 kDa (Fig. 1). The deduced amino acid sequence of GIV-64L was aligned with the published sequences of another 10 sequenced iridoviruses. As Fig. 1a illustrates, no putative conserved domain was detected, but the identical percentage of the amino acids sequences of GIV-64L with SGIV-93L was over 97% (Table 1). The abbreviations and accession numbers of the 10 published sequences of iridovirus are listed in Table 1. To understand the position of GIV-64L in the evolutionary history, a neighbour-joining phylogenetic tree was constructed with all amino acid sequences of the 10 published sequences and GIV-64L. As shown in Fig. 1b, GenBank searches revealed that GIV-64L homologues were present only in the members of the Ranavirus. Ó 2015 John Wiley & Sons Ltd
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Expression and purification of recombinant GIV-64L-His protein The full length of GIV-64L was amplified by PCR from GIV genomic DNA (Fig. 2a). Sequence analysis revealed that the PCR product contained the full length of GIV-64L, which was amplified by PCR from GIV genomic DNA and cloned into a pET-23a vector. After transformation into the E. coli XL1-Blue strain, the recombinant plasmid was extracted and identified by restriction enzyme digestion (Fig. 2b) and DNA sequencing (data not shown), and results showed that the recombinant gene was present. Next, for the recombinant gene (GIV-64L-His) expression, the correct plasmid pET23a-GIV-64L was transformed into an E. coli BL21(DE3) strain that can produce T7 RNA polymerase in the presence of IPTG. The recombinant GIV-64L-His protein was expressed successfully in E. coli BL21 (DE3) cells. As shown in Fig. 2c, the band with an expected molecular weight of approximately 50.2 kDa (429 amino acids) was examined by SDS-PAGE in the cell lysate after 24-h induction with 1 mM IPTG when compared with noninduced negative control. Next, the recombinant GIV-64L-His proteins were purified successfully and verified by SDS-PAGE (Fig. 2c). Production and characterization of GIV-64L mAbs Five mice were immunized with GIV-64L-His protein to elicit an antibody response and generate hybridoma cells. The sera of five mice were all responsive to the antigen in Western blotting, but the serum of mouse number-2 showed the highest sensitivity (data not showed) and thus was used in the subsequent experiments. As shown in Fig 3a, recombinant GIV-64L-His and purified grouper iridovirus particle (pGIVP) could be recognized by the mouse number-2 polyclonal antibodies. The result also indicated that GIV 64L was packed into the assembled viral particles (Fig. 3a). Subsequently, GIV-64L mAbs were generated from the mouse number-2; the hybridoma clones were screened by ELISA against GIV-64L-His protein. Of 46 clones, three were positive against GIV-64L-His protein (GIV-64L-mAb-1, OD650 = 0.56; GIV-64L-mAb-17, OD650 = 0.88; GIV64L-mAb-18, OD650 = 0.74), and all of the secreted mAbs belong to the immunoglobulin G1
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Z-Y Chen et al. Mouse mAbs against GIV-64L
(a)
(b)
Figure 1 Multiple amino acid sequence alignment and phylogenetic tree establishment of GIV-64L. (a) Multiple amino acid sequence alignment of GIV-64L with related gene sequences of 10 other iridoviruses. The identical residues in all sequences are marked by a pound symbol under the alignment. (b) Phylogenetic tree analysis of GIV-64L and related proteins in the family Iridoviridae. The phylogenetic tree was constructed using the neighbour-joining method. Branch lengths are proportional to the evolutionary distance between taxa. Grouper iridovirus ORF 64L: GIV 64L; Singapore grouper iridovirus ORF 93L: SGIV 93L; tiger frog virus ORF 23R: TFV 23R; soft-shelled turtle iridovirus ORF 27R: STIV 27R; frog virus 3 ORF 23R: FV3 23R; common midwife toad ranavirus ORF 84L: CMTV 84L; Andrias davidianus ranavirus ORF 87L: ADRV 87L; Ambystoma tigrinum virus ORF 53R: ATV 53R; European catfish virus ORF 75R: ECV 75R; Chinese giant salamander iridovirus ORF 90L: CGSIV 90L; epizootic haematopoietic necrosis virus ORF 55R: EHNV 55R.
isotype (data not showed). As Fig. 3b demonstrates, Western blot showed that these three GIV-64-mAbs all could specifically recognize Ó 2015 John Wiley & Sons Ltd
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pGIVP. Among the mAbs, GIV-64L-mAb-17 hybridoma cells were injected into mice to produce ascites for subsequent studies, from which
Z-Y Chen et al. Mouse mAbs against GIV-64L
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Figure 2 GIV-64L gene isolation, recombinant pET23a-GIV-64L plasmid construction and recombinant GIV-64L-His protein purification. (a) PCR amplification of a GIV-64L 1218-bp fragment from GIV genomic DNA. M: DNA marker; arrowhead indicates position of GIV-64L. (b) Construction of the recombinant plasmid pET23a-GIV-64L. M: DNA marker; pET23a-GIV-64L; recombinant pET23a-GIV-64L expression plasmid; pET23a-GIV-64L + EcoRI + XhoI: recombinant plasmid pET23a-GIV-64L digested with EcoRI and XhoI. Arrowhead indicates position of GIV-64L fragment. (c) GIV-64L-His protein expression and purification. The expressed and purified GIV-64L-His protein was separated by SDS-PAGE. Gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker; non-IPTG: non-IPTG-induced cell lysate; IPTG: IPTG-induced cells lysate; GIV64L-His: purified GIV-64L-His protein. Arrowhead indicates position of GIV-64L-His.
Ó 2015 John Wiley & Sons Ltd
the mAbs were prepared successfully after treatments with saturated ammonium sulphate solution and protein G agarose (data not showed). We analysed the detection sensitivity of the prepared GIV-64L-mAb-17 by Western blot. In the assay, the GIV-64L-His antigen concentration was applied in a serial dilution from 5 lg to 0.01 lg per lane and GIV-64L-mAb-17 (500 lg mL1) was diluted 1:5000 in PBST. Colour signals were visualized with a goat anti-mouse secondary antibody conjugated to AP (1:5000) and NBT/ BCIP substrate. As shown in Fig. 3c, GIV64L-mAb-17 could detect GIV-64L-His antigen with an approximate detection limit dilution of 0.1 lg.
performed on material from GIV-infected GK cells at 1, 3, 6, 12, 18, 24 and 36 hpi. In Fig. 4a, the transcript of GIV-45R(MCP) was detected as early as 12 hpi, whereas the GIV-64L gene fragment was first detected at 18 hpi and a high level of transcription continued until 36 hpi. To verify the nature of the GIV-64L viral gene, the transcription of GIV-64 was assayed in GIV-infected cells in the presence of CHX or AraC. As shown in Fig. 4b, the transcription of GIV-45R(MCP) and GIV-64L was inhibited by CHX and AraC in GIV-infected GK cells at 12, 18, 24 and 36 hpi. The data support that GIV-64L, like GIV-45R (MCP), is a viral late gene in GIV-infected GK cells.
Temporal expression of the GIV-64L gene in GIV-infected GK cells
Temporal expression of GIV-64L protein in GIV-infected GK cells
To study the temporal transcription of GIV-64L in GIV-infected GK cells, RT-PCR was
To study the temporal translation of GIV-64L in GIV-infected GK cells, Western blotting was
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(a) kDa 75 64
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48
48
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35
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5
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Figure 3 Production, screening, purification and affinity assay of mouse antibody against GIV-64L. (a) Production of mouse polyclonal serum against GIV-64L by Western blotting analysis. M: prestained protein marker; cell lysate: cell lysate of expression protein of pET23a empty vector; GIV-64L-His: purified recombinant GIV-64L-His protein; pGIVP: purified grouper iridovirus particle. (b) Screening of three positive hybridoma cells by Western blotting analysis using purified recombinant GIV-64L-His protein (0.5 lg/per lane) as antigen. M, prestained protein marker. (c) Titration of GIV-64L-mAb-17. Western blotting was performed to titrate GIV-64L-mAb-17 against GIV-64L-His protein at the quantity ranging from 0.01 to 5 lg per lane. GIV-64L-mAb-17 (500 lg mL1) was diluted 1:5000 in PBST.
performed on material from GIV-infected GK cells at 1, 3, 6, 12, 18, 24 and 36 hpi (Fig. 5a). As shown in Fig. 5b, two late genes, GIV-55L and GIV-45R(MCP), were first detected at 12 hpi by Western blotting. Comparatively, the signal of GIV-55L was much fainter than GIV-45R(MCP). In GIV-64L protein expression, no signal was detected until 24 hpi by Western blotting assay. Overall, results depicted in Figs 4 & 5 demonstrate that GIV-64L might be a viral late gene and that GIV-64L-mAb-17 can be used as an effective detection tool in Western blotting. Subcellular localization of GIV-64L in GIV-infected GK cells To study the subcellular localization of GIV-64L, GIV-64L-mAb-17 was used in an immunofluorescence assay to examine the intracellular localization of GIV-64L in GIV-infected GK cells. To study the subcellular localization of GIV-64L, GIV-64L-mAb-17 was used in an immunofluorescence assay to locate GIV-64L in GIV-infected GK cells. As shown in Fig. 6, no significant fluorescent signal change was observed in GIVinfected cells compared with mock infection at 12 hpi, but the green fluorescent signal of GIV64L was observed in almost all GIV-infected cells Ó 2015 John Wiley & Sons Ltd
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at 24 hpi, which is consistent with the Western blot result. Interestingly, the green fluorescence signals of GIV-64L were distributed mainly in the cytoplasm. This result demonstrates that GIV64L-mAb-17, in addition to serving as an effective detection tool in Western blotting, can function in immunofluorescence assays. Discussion
Grouper (Epinephelus spp.) is an economically important fish species in Asia, and GIV is one of the most important viral pathogens, having caused significant economic losses in the grouper aquaculture industry (Chi 1997; Lai et al. 2000, 2001). Although the sequence of the virus genome is now known, understanding of the molecular mechanisms underlying the pathogenicity of iridovirus is still limited, mainly because of insufficient gene characterization. In this study, a late gene encoded by GIV-64L was investigated. GIV-64L is 1215 bp in length and can encode peptide that contains 405 amino acids and has a molecular mass of 47.5 kDa. Compared with the homologues from other iridovirus isolates, our results showed significant identity only to members of the Ranavirus genus (Fig. 1). The evolutionary relationship of GIV-64L with other homologues
Z-Y Chen et al. Mouse mAbs against GIV-64L
Journal of Fish Diseases 2016, 39, 129–141
(a)
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GIV-64L GIV-45R (MCP) β-Actin
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GIV-64L + CHX GIV-45R (MCP) + CHX GIV-64L + AraC GIV-45R (MCP) + AraC Figure 4 Temporal expression pattern of GIV-64L transcription compared with GIV-45R(MCP) was measured by RT-PCR during GIV infection of GK cells. (a) GK cells were infected with GIV at MOI = 5, and transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. (b) GK cells were infected with GIV at MOI = 5 with the presence of CHX or AraC, and the transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. Grouper iridovirus ORF 64L: (GIV64L); grouper iridovirus ORF 45R (major capsid protein): GIV-45R(MCP); cycloheximide: CHX; cytosine arabinoside: AraC.
(a)
kDa M
(b)
0h
0 h 3 h 6 h 12 h 18 h 24 h 36 h
3h
6h
12 h
18 h
24 h
36 h
GIV-64L 75 64 48
GIV-55L
35 GIV-45R(MCP) 25 17
β-Actin
11 Figure 5 Temporal expression pattern of GIV-64L, GIV-45R(MCP) and GIV-55L proteins. (a) GK cells were infected with GIV at MOI = 5, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. Cell lysates were separated by SDS-PAGE. The gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker. (b) GK cells were infected with GIV at MOI = 5, and translation of GIV-64L, GIV-45R(MCP) and GIV-55L was measured by Western blotting analysis, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. GIV-64L protein was detected with GIV-64L-mAb-17. Grouper iridovirus ORF 55L: GIV-55L.
was consistent with the position of GIV in iridovirus evolution. Monoclonal antibodies are a good tool for detecting and characterizing viral infection (Patil et al. 2013; Zhang et al. 2013). In recent years, mAb technology has had an important impact on aquaculture disease diagnostics (Lai et al. 2001, Ó 2015 John Wiley & Sons Ltd
137
2002; Shi et al. 2003; Cote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In our previous study, GIV-45R(MCP), GIV-55L and GIV-97L mAbs were produced and used successfully to verify the detection and subcellular localization of target viral proteins in GIV-infected GK cells. In
Z-Y Chen et al. Mouse mAbs against GIV-64L
Journal of Fish Diseases 2016, 39, 129–141
Mock infection
12 h p.i.
24 h p.i.
DAPI
FITC
Merge
Figure 6 Localization assay of GIV-64L in GIV-infected GK cells. Subcellular localization of GIV-64L was analysed using GIV64L-mAb-17 against GIV-64L by immunofluorescence assay during GIV infection of GK cells, respectively, at 0, 12 and 24 hpi. The nucleus was counter-stained by DAPI. The fluorescent signal was examined under a fluorescence microscope (Olympus IX81 Confocal Microscope, Japan).
this study, we developed and characterized three mouse mAbs (GIV-64L-mAb-1, GIV-64L-mAb17 and GIV-64L-mAb-18) that are specific to the GIV-64L protein of GIV. Further analyses demonstrated that these mAbs could serve as tools for investigating the expression and potential role of GIV-64L during GIV infection. Among the mAbs, GIV-64L-mAb-17 was the most reactive in ELISA, so we prepared GIV-64L-mAb-17 in a large quantity for subsequent studies. It proved to be an effective reagent for detecting the temporal expression and intracellular localization of GIV-64L protein during GIV infection of GK cells (Fig. 3). Generally, transcription of iridovirus genes can be classified into immediate early (IE), early and late genes according to their temporal synthesis (Williams 1996). By definition, the expression of IE genes relies solely on host proteins, which are present before viral DNA replication. The IE proteins are essential for the viral life cycle (Willis & Ó 2015 John Wiley & Sons Ltd
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Granoff 1985; Xia et al. 2010), and their 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; Holley-Guthrie et al. 1990; Williams et al. 2005; Huang et al. 2011). Expression of early genes, dependent on the preceding expression of IE genes, mainly encodes enzymes required for viral DNA synthesis and for regulating the expression of late genes. On the other hand, viral late genes are expressed after the onset of DNA replication and encode mainly structural or envelope proteins of viral particles and often are selected as vaccine candidate targets (Ebrahimi et al. 2003; Lua et al. 2005; Sanchez-Paz 2010). In our previous study, GIV-45R(MCP) and GIV55L genes were demonstrated to be late genes in GIV infection of GK cells (Hu et al. 2014; Lin et al. 2014). In the present study, the temporal expression analysis of GIV-64L was compared with that of
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GIV-45R(MCP) by RT-PCR 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. Our data showed that transcription of GIV-64L and GIV-45R(MCP) was inhibited by CHX and AraC. In addition, GIV-45R(MCP) was detected at 12 h, but GIV-64L was detected at 18 h after GIV infection (Fig. 4). The GIV-64L sequence was significantly similar to that of SGIV-93L (Singapore grouper iridovirus-93L). In a previous study, SGIV-93L was classified as a late gene by DNA microarray analysis (Chen et al. 2006) and was predicted to belong to a viral envelope protein of SGIV by proteomic analysis (Zhou et al. 2011). In the temporal translation assay of GIV64L, our data indicate that two late genes, GIV45R(MCP) and GIV-55L, were detected at 12 h, but GIV-64L was detected until 24 h after GIV infection (Fig. 4b). Overall, these data support that GIV-64L is a later gene than GIV-45R (MCP) and GIV-55L during viral replication and finally is packed into the whole virus particle. Some GIV genes localized in GIV-infected GK cells and previously characterized in our laboratory, including GIV-45R(MCP) and GIV-97L, were found to be in both the cytoplasm and nucleus, whereas GIV-55L was found only in the cytoplasm during GIV infection of GK cells. In the present study, the immunofluorescence assay with GIV64L-mAb-17 showed that GIV-64L, like GIV55L, localized only in the cytoplasm and co-localization with the virus factory during GIV infection of GK cells (Wan et al. 2010; Hu et al. 2014). In conclusion, we have cloned and identified the GIV-64L gene of GIV. GenBank searches revealed that GIV-64L homologues were present only in the members of Ranavirus. Mouse mAbs specific to GIV-64L were produced, and we have shown that these mAbs could also be valuable tools in the study of the biological function and pathological significance of GIV-64L during viral infection. Finally, GIV-64L was identified to be a very late gene and to encode a protein localized in the cytoplasm of infected cells before finally being packed into the whole virus particle. Acknowledgements This study was supported by Grant no. NSC-1022815-C-197-008-B from the National Science Council, Taiwan. Ó 2015 John Wiley & Sons Ltd
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References Aamelfot M., Weli S.C., Dale O.B., Koppang E.O. & Falk K. (2013) Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor. Journal of Anatomy 222, 547–557. Buisson M., Manet E., Trescol-Biemont M.C., Gruffat H., Durand B. & Sergeant A. (1989) The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R. Journal of Virology 63, 5276–5284. Chen L.M., Wang F., Song W. & Hew C.L. (2006) Temporal and differential gene expression of Singapore grouper iridovirus. The Journal of General Virology 87, 2907–2915. Chi S.C. (1997) The investigation of viral disease among cultured groupers in southern Taiwan. COA Fisheries Series No. 61, Reports on Fish Disease Research 18, 59–69. Chinchar V.G., Essbauer S., He J.G., Hyatt A., Miyazaki T., Seligy V. & Williams T. (2005) Family Iridoviridae. In: Virus Taxonomy: XIII Report of the International Committee on Taxonomy of Viruses (ed. by C.M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger & L.A. Ball), pp. 163–175. Elsevier, London. Chiou P.P., Chen Y.C. & Lai Y.S. (2009) Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus. Journal of Fish Diseases 32, 997–1005. Chua F.H.C., Ng M.L., Ng K.L., Loo J.J. & Wee J.Y. (1994) Investigation of outbreaks of a novel disease, ‘Sleepy Grouper Disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. Journal of Fish Diseases 17, 417–427. Cote I., Poulos B.T., Redman R.M. & Lightner D.V. (2009) Development and characterization of a monoclonal antibody against Taura syndrome virus. Journal of Fish Diseases 32, 989–996. Ebrahimi B., Dutia B.M., Roberts K.L., Garcia-Ramirez J.J., Dickinson P., Stewart J.P., Ghazal P., Roy D.J. & Nash A.A. (2003) Transcriptome profile of murine gammaherpesvirus-68 lytic infection. Journal of General Virology 84, 99–109. He J.G., Wang S.P., Zeng K., Huang Z.J. & Chan S.M. (2000) Systemic disease caused by an iridovirus-like agent in cultured mandarinfish, Siniperca chuatsi (Basilewsky), in China. Journal of Fish Diseases 23, 219–222. Holley-Guthrie E.A., Quinlivan E.B., Mar E.C. & Kenney S. (1990) The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. Journal of Virology 64, 3753–3759. Hou C.L., Cao Y., Xie R.H., Wang Y.Z. & Du H.H. (2011) Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus. Virologica Sinica 26, 260–266. Hu S.L., Liou C.J., Cheng Y.H., Yiu J.C., Chiou P.P. & Lai Y.S. (2014) Development and characterization of two
Journal of Fish Diseases 2016, 39, 129–141
monoclonal antibodies against grouper iridovirus 55L and 97L proteins. Journal of Fish Diseases. Epub ahead of print. Huang X., Huang Y., Ouyang Z., Cai J., Yan Y. & Qin Q. (2011) Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells. The Journal of General Virology 92, 1292–1301. Lai Y.S., Murali S., Ju H.Y., Wu M.F., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2000) Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus. Journal of Fish Diseases 23, 379–388. Lai Y.S., Chiu H.C., Murali S., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2001) In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus Awoara (Temminck & Schlegel). Journal of Fish Diseases 24, 237–244. Lai Y.S., John J.A., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2002) In vitro efficiency of intra- and extracellular immunization with mouse anti-YGNNV antibody against yellow grouper nervous necrosis virus. Vaccine 20, 3221–3229. Lai Y.S., John J.A., Lin C.H., Guo I.C., Chen S.C., Fang K., Lin C.H. & Chang C.Y. (2003) Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses. Journal of Fish Diseases 26, 31–42. Lai Y.S., Chiou P.P., Chen W.J., Chen Y.C., Chen C.W., Chiu I.S., Chen S.D., Cheng Y.H. & Chang C.Y. (2008) Characterization of apoptosis induced by grouper iridovirus in two newly established cell lines from barramundi, Lates calcarifer (Bloch). Journal of Fish Diseases 31, 825–834.
tiger shrimp Penaeus monodon. Journal of Fish Diseases 36, 753–762. Pham P.H., Lai Y.S., Lee F.F., Bols N.C. & Chiou P.P. (2012) Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three non-host species. Virus Research 167, 16–25. Qin Q.W., Chang S.F., Ngoh-Lim G.H., Gibson-Kueh S., Shi C. & Lam T.J. (2003) Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina. Diseases of Aquatic Organisms 53, 1–9. Reed L.J. & Muench H. (1938) A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 493–497. Sanchez-Paz A. (2010) White spot syndrome virus: an overview on an emergent concern. Veterinary Research 41, 43. Schuh J.C.L. & Shirley I.G. (1990) Viral hematopoietic necrosis in an angelfish (Pterophyllum scalare). Journal of Zoo and Wildlife Medicine 21, 95–98. Shi C., Wei Q., Gin K.Y. & Lam T.J. (2003) Production and characterization of monoclonal antibodies to a grouper iridovirus. Journal of Virological Methods 107, 147–154. Siriwattanarat R., Longyant S., Chaivisuthangkura P., Wangman P., Vaniksampanna A. & Sithigorngul P. (2013) Improvement of immunodetection of white spot syndrome virus using a monoclonal antibody specific for heterologously expressed icp11. Archives of Virology 158, 967–979. Ting J.W., Wu M.F., Tsai C.T., Lin C.C., Guo I.C. & Chang C.Y. (2004) Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase. The Journal of General Virology 85, 2883–2892.
Langdon J.S. & Humphrey J.D. (1987) Epizootic haematopoietic necrosis, a new viral disease in redfin perch, Perca fluviatilis L., in Australia. Journal of Fish Diseases 10, 289–297.
Tsai C.T., Ting J.W., Wu M.H., Wu M.F., Guo I.C. & Chang C.Y. (2005) Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses. Journal of Virology 79, 2010–2023.
Lin H.Y., Liou C.J., Cheng Y.H., Hsu H.C., Yiu J.C., Chiou P.P. & Lai Y.S. (2014) Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. Journal of Virological Methods 205, 31–37.
Wan Q.J., Gong J., Huang X.H., Huang Y.H., Zhou S., Ou-Yang Z.L., Cao J.H., Ye L.L. & Qin Q.W. (2010) Identification and characterization of a novel capsid protein encoded by Singapore grouper iridovirus ORF038L. Archives of Virology 155, 351–359.
Lua D.T., Yasuike M., Hirono I. & Aoki T. (2005) Transcription program of red sea bream iridovirus as revealed by DNA microarrays. Journal of Virology 79, 15151–15164. Murali S., Wu M.F., Guo I.C., Chen S.C., Yang H.W. & Chang C.Y. (2002) Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel). Journal of Fish Diseases 25, 91–100. Patil R., Shankar K.M., Kumar B.T., Kulkarni A., Patil P. & Moger N. (2013) Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black
Ó 2015 John Wiley & Sons Ltd
Z-Y Chen et al. Mouse mAbs against GIV-64L
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Williams T. (1996) The iridoviruses. Advances in Virus Research 46, 345–412. Williams T., Barbosa-Solomieu V. & Chinchar V.G. (2005) A decade of advances in iridovirus research. Advances in Virus Research 65, 173–248. Willis D.B. & Granoff A. (1985) trans activation of an immediate-early frog virus 3 promoter by a virion protein. Journal of Virology 56, 495–501. Wu M.S., Chen C.W., Liu Y.C., Huang H.H., Lin C.H., Tzeng C.S. & Chang C.Y. (2012) Transcriptional analysis of orange-spotted grouper reacting to experimental grouper iridovirus infection. Developmental and Comparative Immunology 37, 233–242.
Journal of Fish Diseases 2016, 39, 129–141
Ó 2015 John Wiley & Sons Ltd
Z-Y Chen et al. Mouse mAbs against GIV-64L
Xia L., Liang H., Huang Y., Ou-Yang Z. & Qin Q. (2010) Identification and characterization of Singapore grouper iridovirus (SGIV) ORF162L, an immediate-early gene involved in cell growth control and viral replication. Virus Research 147, 30–39.
Zhou S., Wan Q., Huang Y., Huang X., Cao J., Ye L., Lim T.-K., Lin Q. & Qin Q. (2011) Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088. Proteomics 11, 2236–2248.
Zhang Z., Li X., Yi W., Li S., Hu C. & Chen A. (2013) A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection. Clinical Biochemistry 46, 1105–1110.
Received: 14 July 2014 Revision received: 3 November 2014 Accepted: 4 November 2014
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Production and characterization of a monoclonal antibody against a late gene encoded by grouper iridovirus 64L Z-Y Chen1, P P Chiou1,2, C-J Liou3,4 and Y-S Lai1 1 2 3 4
Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan
Abstract
Viral envelope proteins play important roles in viral infection and assembly. The grouper iridovirus ORF 64L (GIV-64L) was predicted to encode an envelope protein and was conserved in all sequenced Ranaviruses. In this study, the complete nucleotide sequence of the GIV-64L gene (1215 bp) was cloned into the isopropyl b-D-1thiogalactopyranoside (IPTG) induction prokaryotic expression vector pET23a. The approximately 50.2 kDa recombinant GIV-64L-His protein was induced, purified and used as an immunogen to immunize BALB/c mice. Three monoclonal antibodies (mAbs), all IgG1 class antibodies against GIV-64L protein, were produced by enzyme-linked immunosorbent assay. Reverse transcription polymerase chain reaction analyses revealed GIV-64L to be a late gene when expressed in grouper kidney cells during GIV infection with cycloheximide (an inhibitor of protein synthesis) or cytosine arabinoside (an inhibitor of DNA synthesis) present. Finally, one of the established mAbs, GIV-64L-mAb-17, was used in Western blotting and an immunofluorescence assay, which showed that GIV-64L protein was expressed at 24 h post-infection and localized only in the cytoplasm in GIV-infected cells, packed into a whole virus particle. The presently characterized GIV-64L mAbs should have 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]) Ó 2015 John Wiley & Sons Ltd
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widespread applications in GIV immunodiagnostics and other research, and these results should offer important insights into the pathogenesis of GIV. Keywords: GIV-64L, Grouper iridovirus, immunofluorescence, monoclonal antibody. Introduction
Grouper (Epinephelus spp.) is one of the most economically important cultured marine fish in many Asian countries. Viral disease is a major constraint in the hatchery production of grouper, and iridoviruses are among the most important viral pathogens in these fish, particularly at the fry and fingerling stages. Iridoviruses are large icosahedral viruses containing a linear double-stranded DNA packed in a viral particle with diameters ranging from 120 to 350 nm (Williams 1996). The Iridoviridae family is classified into five genera— Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus and Megalocytivirus—whose members can infect only ectothermic vertebrates and invertebrates (Chinchar et al. 2005; Williams, BarbosaSolomieu & Chinchar 2005). Grouper infected with iridovirus might show darkened body colour, pale gills and an enlarged spleen, and often become lethargic with low appetite (Langdon & Humphrey 1987; Schuh & Shirley 1990; Chua et al. 1994; He et al. 2000; Lai et al. 2000). Microscopic pathological signs might include enlargement of cells and necrosis of renal and splenic hematopoietic tissues (Qin et al. 2003).
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Grouper iridovirus (GIV), a member of the Ranavirus genus that was originally isolated from southern Taiwan, has caused significant economic losses in the grouper aquaculture industry for years (Murali et al. 2002; Tsai et al. 2005). GIVsusceptible cell lines from yellow grouper have been established (Lai et al. 2000, 2003), and the complete genome of GIV has been sequenced and consists of 139 793 bp with a 49% G + C content. The genome is predicted to encode 120 open reading frames (ORFs), ranging in size from 62 to 1268 amino acids (Tsai et al. 2005). In recent years, although the GIV genome sequence, host cellular gene expression (Wu et al. 2012) and apoptosis characteristics have been investigated (Lai et al. 2008; Chiou, Chen & Lai 2009; Pham et al. 2012), the understanding of the molecular aspects of GIV pathogenicity is still limited. Four GIV genes have been identified and characterized: GIV-45R (major capsid protein) (Lin et al. 2014), GIV-49L (predicted function and/or similarity: purine nucleoside phosphorylase) (Ting et al. 2004), GIV-55L (predicted function and/or similarity: RNase III) and GIV-97L (predicted function and/or similarity: NTPase–helicase) (Hu et al. 2014). Mouse monoclonal antibodies (mAbs) are efficient tools for detecting and characterizing viral pathogens. In recent years, mAb technology has had an important effect on aquaculture disease diagnosis (Lai et al. 2001, 2002; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). To understand the molecular mechanism of GIV pathogenesis, the expression pattern and assay tools of viral genes must be developed and evaluated. Viral envelope proteins are critical to virus infection and virus–host interaction, and their specific antibodies can serve as a valuable tool for virus detection. The GIV-64L was predicated in silico to encode an envelope protein, thus could be a good candidate target of immunological detection of the presence of the virus. In this study, we cloned the GIV-64L gene from GIV genomic DNA and produced mouse mAbs specific to GIV-64L. This investigation marks the first examination of GIV-64L gene expression and subcellular protein location during GIV infection in vitro, and these specific mAbs are a valuable tool for GIV diagnostics and studies on GIV pathogenesis. Ó 2015 John Wiley & Sons Ltd
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Materials and methods
Cells and viruses Grouper kidney (GK) cells previously established and characterized from the kidney mass of grouper were used in this study (Lai et al. 2000). The propagation and purification of GIV was conducted in GK cells as described previously (Lai et al. 2000). GK cells (107) growth in a 175-cm2 flask was infected with GIV at a multiplicity of infection (MOI) of 0.01. The virus was then allowed to multiply for 7 days at 28 °C. The culture mediums of 10 flasks were collected and centrifuged at 2000 9 g for 30 min at 4 °C. The supernatant and pellet were collected separately. The supernatant was mixed with polyethylene glycol (PEG, Sigma), stirred at 4 °C overnight and then centrifuged at 4000 9 g for 1 h at 4 °C. The pellet was resuspended in 2 mL of TNE buffer (50 mM Tris–HCl, 1 mM EDTA (ethylene diamine tetraacetic acid), 100 mM NaCl, pH 7.3), mixed with an equal volume of Freon (1,1,2-trichlorotrifluoroethane, Sigma) and centrifuged at 1800 9 g for 10 min. The pellet (cell-associated virus) was also extracted with Freon in the same way. Aqueous solutions of both virus samples were collected and combined, layered over a three-step caesium chloride gradient (4 mL 40%, 3 mL 30%, 2 mL 20%) and centrifuged at 13 000 9 g for 16 h at 4 °C. The virus band was collected and diluted with 5 mL TNE buffer and recentrifuged at 23 000 9 g for 2 h at 4 °C. The purified virus pellet was resuspended in 1 mL of TNE buffer and used for all analyses. The virus titre was calculated by the TCID50 method, described in Reed & Muench (1938). Computer-assisted analysis for protein alignment and phylogenetic tree generation GIV-64L homology analyses were performed using the BLAST network server of the National Center for Biotechnology Information. The multiple alignment of the GIV-64L amino acid sequence with those of another 10 sequenced iridoviruses was edited and performed using the GeneDoc program. The phylogenetic tree was generated using MEGA 6 software. The detail information for the other iridoviruses using in the blast analysis and phylogenetic tree was listed in Table 1.
Z-Y Chen et al. Mouse mAbs against GIV-64L
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pET23a-GIV-64L plasmid construction The full length of GIV-64L was isolated from GIV genomic DNA by polymerase chain reaction (PCR) using a pair of primers: GIV-64L-F/64L-R (Table 1). PCR was carried out under the following conditions: 10 min at 95 °C for one cycle; 1 min at 95 °C, 1 min at 56 °C and 1 min at 72 °C for 35 cycles; and 10 min at 72 °C for one cycle. The amplified fragment of GIV-64L was cloned into the pET23a (Novagen) expression vector using T4 DNA ligase (Roche). These constructs were transformed into Escherichia coli (E. coli) XL1-Blue, and the transformation was confirmed by restriction enzyme digestion and DNA sequencing. Preparation of recombinant GIV-64L-His protein After DNA sequencing, the recombinant fusion construct, pET23a-GIV-64L expression vector, was transformed into E. coli BL21(DE3)-competent cells, and recombinant GIV-64L-histidine tag (GIV-64L-His) protein was induced by 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) (Sigma) for 24 h at 37 °C. Thereafter, cells were cooled on ice for 30 min and harvested by centrifugation (4000 9 g, 20 min). 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 insoluble debris was removed by centrifugation at 10 000 9 g for 30 min, and the supernatant was collected and loaded onto a Ni-NTA resin affinity column (GEHealthcare). The column was successively washed with lysis buffer A containing 1, 10, 20 and 200 mM imidazole, respectively. The eluted fractions were
collected and analysed by 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie brilliant blue R-250. 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) until the imidazole was removed and stored at 20 °C until use. Immunization and antibody production Five female (8 weeks old) BALB/c mice (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used for immunization. For the first-time injection, each mouse was injected subcutaneously with 200 lL of emulsion containing 100 lg of GIV-64L-His and Freund’s complete adjuvant (Sigma). Then, the mouse was boosted with same volume of emulsion mixed 1:1 with incomplete Freund’s adjuvant (Sigma) at 2 weeks and again at 4 weeks. The mouse antiserum was collected every week to assess immunity by Western blotting and enzymelinked immunosorbent assay (ELISA). All the animal experiments were performed in accordance with the guidelines of the ethics review committee for animal experiments at National Ilan University. The protocols for mouse immunization and cell fusion were similar to those previously described (Hu et al. 2014). The selection of hybridoma clones producing mAbs specific against GIV-64L proteins was accomplished by ELISA and Western blotting. ELISA assay For ELISA, the ELISA 96-well plates (Nunc) were coated with 0.1 lg/well of recombinant GIV-64L-
Table 1 GenBank accession numbers of GIV-64L homologues used in this study
Ó 2015 John Wiley & Sons Ltd
Virus names
ORF
Accession no.
Identity (%)
Length a.a.
Grouper iridovirus (GIV) Singapore grouper iridovirus (SGIV) Tiger frog virus (TFV) Soft-shelled turtle iridovirus (SUV) Frog virus 3 (FV3) Common midwife toad ranavirus (CMTV) Andrias davidianus ranavirus (ADRV) Ambystoma tigrinum virus (ATV) European catfish virus (ECV) Chinese giant salamander iridovirus (CGSIV) Epizootic haematopoietic necrosis virus (EIINV)
64L 93L 23R 27R 23R 84L 87L 53R 75R 90L 55R
AAV91080 YP164188 ABB92288 ACF42246 YP031601 AFA44990 AGV20618 YP003826 YP006347667 AHA80935 ACQ25245
97 23 23 23 23 23 22 22 23 23
405 405 382 382 382 382 382 382 382 365 353
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(a)
C
1h
3h
6h
12 h
18 h
24 h
36 h
C
1h
3h
6h
12 h
18 h
24 h
36 h
GIV-64L GIV-45R (MCP) β-Actin
(b)
GIV-64L + CHX GIV-45R (MCP) + CHX GIV-64L + AraC GIV-45R (MCP) + AraC Figure 4 Temporal expression pattern of GIV-64L transcription compared with GIV-45R(MCP) was measured by RT-PCR during GIV infection of GK cells. (a) GK cells were infected with GIV at MOI = 5, and transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. (b) GK cells were infected with GIV at MOI = 5 with the presence of CHX or AraC, and the transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. Grouper iridovirus ORF 64L: (GIV64L); grouper iridovirus ORF 45R (major capsid protein): GIV-45R(MCP); cycloheximide: CHX; cytosine arabinoside: AraC.
(a)
kDa M
(b)
0h
0 h 3 h 6 h 12 h 18 h 24 h 36 h
3h
6h
12 h
18 h
24 h
36 h
GIV-64L 75 64 48
GIV-55L
35 GIV-45R(MCP) 25 17
β-Actin
11 Figure 5 Temporal expression pattern of GIV-64L, GIV-45R(MCP) and GIV-55L proteins. (a) GK cells were infected with GIV at MOI = 5, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. Cell lysates were separated by SDS-PAGE. The gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker. (b) GK cells were infected with GIV at MOI = 5, and translation of GIV-64L, GIV-45R(MCP) and GIV-55L was measured by Western blotting analysis, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. GIV-64L protein was detected with GIV-64L-mAb-17. Grouper iridovirus ORF 55L: GIV-55L.
was consistent with the position of GIV in iridovirus evolution. Monoclonal antibodies are a good tool for detecting and characterizing viral infection (Patil et al. 2013; Zhang et al. 2013). In recent years, mAb technology has had an important impact on aquaculture disease diagnostics (Lai et al. 2001, Ó 2015 John Wiley & Sons Ltd
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2002; Shi et al. 2003; Cote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In our previous study, GIV-45R(MCP), GIV-55L and GIV-97L mAbs were produced and used successfully to verify the detection and subcellular localization of target viral proteins in GIV-infected GK cells. In
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immunoglobulins (400 lg mL1, 1:5000) (Santa Cruz) at 37 °C for 1 h. Finally, the colour band was developed in the dark using the substrates 4-nitro-blue tetrazolium chloride/5-bromo-4chloro-3-indolyl phosphate (NBT/BCIP) (Sigma). Immunofluorescence assay To analyse the location of the GIV-64L protein, GK cells were grown on a coverslip in a 12well plate and infected with GIV at a MOI = 5. At 0, 24 and 30 hpi, cells were fixed with 2% paraformaldehyde/0.1% Triton X-100 for 30 min at 37 °C. After removal of fixative, the coverslip was blocked for 30 min at 37 °C by bovine serum albumin. Cells were incubated with GIV-64L-mAb-17 (500 lg mL1, 1:100) for 1 h at 37 °C. After three washes with PBS, the fluorescein-labelled goat anti-mouse immunoglobulin G (H+L) (500 lg mL1, 1:50) (KPL) was added into the well and incubated for 1 h at 37 °C. Cellular and GIV DNA were labelled with 4, 6-diamidino-2-phenylindole (DAPI) for 10 min. The slides were washed with PBS three times, mounted and observed under a confocal fluorescence microscope (Olympus IX81, Japan).
Results
Identification and sequence analysis of GIV-64L GIV-64L (GenBank accession no. AAV91080) is 1215 bp in length, encoding a putative protein of 405 amino acids with a predicted molecular mass of 47.5 kDa (Fig. 1). The deduced amino acid sequence of GIV-64L was aligned with the published sequences of another 10 sequenced iridoviruses. As Fig. 1a illustrates, no putative conserved domain was detected, but the identical percentage of the amino acids sequences of GIV-64L with SGIV-93L was over 97% (Table 1). The abbreviations and accession numbers of the 10 published sequences of iridovirus are listed in Table 1. To understand the position of GIV-64L in the evolutionary history, a neighbour-joining phylogenetic tree was constructed with all amino acid sequences of the 10 published sequences and GIV-64L. As shown in Fig. 1b, GenBank searches revealed that GIV-64L homologues were present only in the members of the Ranavirus. Ó 2015 John Wiley & Sons Ltd
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Expression and purification of recombinant GIV-64L-His protein The full length of GIV-64L was amplified by PCR from GIV genomic DNA (Fig. 2a). Sequence analysis revealed that the PCR product contained the full length of GIV-64L, which was amplified by PCR from GIV genomic DNA and cloned into a pET-23a vector. After transformation into the E. coli XL1-Blue strain, the recombinant plasmid was extracted and identified by restriction enzyme digestion (Fig. 2b) and DNA sequencing (data not shown), and results showed that the recombinant gene was present. Next, for the recombinant gene (GIV-64L-His) expression, the correct plasmid pET23a-GIV-64L was transformed into an E. coli BL21(DE3) strain that can produce T7 RNA polymerase in the presence of IPTG. The recombinant GIV-64L-His protein was expressed successfully in E. coli BL21 (DE3) cells. As shown in Fig. 2c, the band with an expected molecular weight of approximately 50.2 kDa (429 amino acids) was examined by SDS-PAGE in the cell lysate after 24-h induction with 1 mM IPTG when compared with noninduced negative control. Next, the recombinant GIV-64L-His proteins were purified successfully and verified by SDS-PAGE (Fig. 2c). Production and characterization of GIV-64L mAbs Five mice were immunized with GIV-64L-His protein to elicit an antibody response and generate hybridoma cells. The sera of five mice were all responsive to the antigen in Western blotting, but the serum of mouse number-2 showed the highest sensitivity (data not showed) and thus was used in the subsequent experiments. As shown in Fig 3a, recombinant GIV-64L-His and purified grouper iridovirus particle (pGIVP) could be recognized by the mouse number-2 polyclonal antibodies. The result also indicated that GIV 64L was packed into the assembled viral particles (Fig. 3a). Subsequently, GIV-64L mAbs were generated from the mouse number-2; the hybridoma clones were screened by ELISA against GIV-64L-His protein. Of 46 clones, three were positive against GIV-64L-His protein (GIV-64L-mAb-1, OD650 = 0.56; GIV-64L-mAb-17, OD650 = 0.88; GIV64L-mAb-18, OD650 = 0.74), and all of the secreted mAbs belong to the immunoglobulin G1
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(a)
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Figure 1 Multiple amino acid sequence alignment and phylogenetic tree establishment of GIV-64L. (a) Multiple amino acid sequence alignment of GIV-64L with related gene sequences of 10 other iridoviruses. The identical residues in all sequences are marked by a pound symbol under the alignment. (b) Phylogenetic tree analysis of GIV-64L and related proteins in the family Iridoviridae. The phylogenetic tree was constructed using the neighbour-joining method. Branch lengths are proportional to the evolutionary distance between taxa. Grouper iridovirus ORF 64L: GIV 64L; Singapore grouper iridovirus ORF 93L: SGIV 93L; tiger frog virus ORF 23R: TFV 23R; soft-shelled turtle iridovirus ORF 27R: STIV 27R; frog virus 3 ORF 23R: FV3 23R; common midwife toad ranavirus ORF 84L: CMTV 84L; Andrias davidianus ranavirus ORF 87L: ADRV 87L; Ambystoma tigrinum virus ORF 53R: ATV 53R; European catfish virus ORF 75R: ECV 75R; Chinese giant salamander iridovirus ORF 90L: CGSIV 90L; epizootic haematopoietic necrosis virus ORF 55R: EHNV 55R.
isotype (data not showed). As Fig. 3b demonstrates, Western blot showed that these three GIV-64-mAbs all could specifically recognize Ó 2015 John Wiley & Sons Ltd
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pGIVP. Among the mAbs, GIV-64L-mAb-17 hybridoma cells were injected into mice to produce ascites for subsequent studies, from which
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Figure 2 GIV-64L gene isolation, recombinant pET23a-GIV-64L plasmid construction and recombinant GIV-64L-His protein purification. (a) PCR amplification of a GIV-64L 1218-bp fragment from GIV genomic DNA. M: DNA marker; arrowhead indicates position of GIV-64L. (b) Construction of the recombinant plasmid pET23a-GIV-64L. M: DNA marker; pET23a-GIV-64L; recombinant pET23a-GIV-64L expression plasmid; pET23a-GIV-64L + EcoRI + XhoI: recombinant plasmid pET23a-GIV-64L digested with EcoRI and XhoI. Arrowhead indicates position of GIV-64L fragment. (c) GIV-64L-His protein expression and purification. The expressed and purified GIV-64L-His protein was separated by SDS-PAGE. Gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker; non-IPTG: non-IPTG-induced cell lysate; IPTG: IPTG-induced cells lysate; GIV64L-His: purified GIV-64L-His protein. Arrowhead indicates position of GIV-64L-His.
Ó 2015 John Wiley & Sons Ltd
the mAbs were prepared successfully after treatments with saturated ammonium sulphate solution and protein G agarose (data not showed). We analysed the detection sensitivity of the prepared GIV-64L-mAb-17 by Western blot. In the assay, the GIV-64L-His antigen concentration was applied in a serial dilution from 5 lg to 0.01 lg per lane and GIV-64L-mAb-17 (500 lg mL1) was diluted 1:5000 in PBST. Colour signals were visualized with a goat anti-mouse secondary antibody conjugated to AP (1:5000) and NBT/ BCIP substrate. As shown in Fig. 3c, GIV64L-mAb-17 could detect GIV-64L-His antigen with an approximate detection limit dilution of 0.1 lg.
performed on material from GIV-infected GK cells at 1, 3, 6, 12, 18, 24 and 36 hpi. In Fig. 4a, the transcript of GIV-45R(MCP) was detected as early as 12 hpi, whereas the GIV-64L gene fragment was first detected at 18 hpi and a high level of transcription continued until 36 hpi. To verify the nature of the GIV-64L viral gene, the transcription of GIV-64 was assayed in GIV-infected cells in the presence of CHX or AraC. As shown in Fig. 4b, the transcription of GIV-45R(MCP) and GIV-64L was inhibited by CHX and AraC in GIV-infected GK cells at 12, 18, 24 and 36 hpi. The data support that GIV-64L, like GIV-45R (MCP), is a viral late gene in GIV-infected GK cells.
Temporal expression of the GIV-64L gene in GIV-infected GK cells
Temporal expression of GIV-64L protein in GIV-infected GK cells
To study the temporal transcription of GIV-64L in GIV-infected GK cells, RT-PCR was
To study the temporal translation of GIV-64L in GIV-infected GK cells, Western blotting was
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Figure 3 Production, screening, purification and affinity assay of mouse antibody against GIV-64L. (a) Production of mouse polyclonal serum against GIV-64L by Western blotting analysis. M: prestained protein marker; cell lysate: cell lysate of expression protein of pET23a empty vector; GIV-64L-His: purified recombinant GIV-64L-His protein; pGIVP: purified grouper iridovirus particle. (b) Screening of three positive hybridoma cells by Western blotting analysis using purified recombinant GIV-64L-His protein (0.5 lg/per lane) as antigen. M, prestained protein marker. (c) Titration of GIV-64L-mAb-17. Western blotting was performed to titrate GIV-64L-mAb-17 against GIV-64L-His protein at the quantity ranging from 0.01 to 5 lg per lane. GIV-64L-mAb-17 (500 lg mL1) was diluted 1:5000 in PBST.
performed on material from GIV-infected GK cells at 1, 3, 6, 12, 18, 24 and 36 hpi (Fig. 5a). As shown in Fig. 5b, two late genes, GIV-55L and GIV-45R(MCP), were first detected at 12 hpi by Western blotting. Comparatively, the signal of GIV-55L was much fainter than GIV-45R(MCP). In GIV-64L protein expression, no signal was detected until 24 hpi by Western blotting assay. Overall, results depicted in Figs 4 & 5 demonstrate that GIV-64L might be a viral late gene and that GIV-64L-mAb-17 can be used as an effective detection tool in Western blotting. Subcellular localization of GIV-64L in GIV-infected GK cells To study the subcellular localization of GIV-64L, GIV-64L-mAb-17 was used in an immunofluorescence assay to examine the intracellular localization of GIV-64L in GIV-infected GK cells. To study the subcellular localization of GIV-64L, GIV-64L-mAb-17 was used in an immunofluorescence assay to locate GIV-64L in GIV-infected GK cells. As shown in Fig. 6, no significant fluorescent signal change was observed in GIVinfected cells compared with mock infection at 12 hpi, but the green fluorescent signal of GIV64L was observed in almost all GIV-infected cells Ó 2015 John Wiley & Sons Ltd
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at 24 hpi, which is consistent with the Western blot result. Interestingly, the green fluorescence signals of GIV-64L were distributed mainly in the cytoplasm. This result demonstrates that GIV64L-mAb-17, in addition to serving as an effective detection tool in Western blotting, can function in immunofluorescence assays. Discussion
Grouper (Epinephelus spp.) is an economically important fish species in Asia, and GIV is one of the most important viral pathogens, having caused significant economic losses in the grouper aquaculture industry (Chi 1997; Lai et al. 2000, 2001). Although the sequence of the virus genome is now known, understanding of the molecular mechanisms underlying the pathogenicity of iridovirus is still limited, mainly because of insufficient gene characterization. In this study, a late gene encoded by GIV-64L was investigated. GIV-64L is 1215 bp in length and can encode peptide that contains 405 amino acids and has a molecular mass of 47.5 kDa. Compared with the homologues from other iridovirus isolates, our results showed significant identity only to members of the Ranavirus genus (Fig. 1). The evolutionary relationship of GIV-64L with other homologues
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GIV-64L + CHX GIV-45R (MCP) + CHX GIV-64L + AraC GIV-45R (MCP) + AraC Figure 4 Temporal expression pattern of GIV-64L transcription compared with GIV-45R(MCP) was measured by RT-PCR during GIV infection of GK cells. (a) GK cells were infected with GIV at MOI = 5, and transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. (b) GK cells were infected with GIV at MOI = 5 with the presence of CHX or AraC, and the transcripts were measured by RT-PCR, respectively, at 1, 3, 6, 12, 18, 24 and 36 hpi. Grouper iridovirus ORF 64L: (GIV64L); grouper iridovirus ORF 45R (major capsid protein): GIV-45R(MCP); cycloheximide: CHX; cytosine arabinoside: AraC.
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11 Figure 5 Temporal expression pattern of GIV-64L, GIV-45R(MCP) and GIV-55L proteins. (a) GK cells were infected with GIV at MOI = 5, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. Cell lysates were separated by SDS-PAGE. The gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker. (b) GK cells were infected with GIV at MOI = 5, and translation of GIV-64L, GIV-45R(MCP) and GIV-55L was measured by Western blotting analysis, respectively, at 0, 3, 6, 12, 18, 24 and 36 hpi. GIV-64L protein was detected with GIV-64L-mAb-17. Grouper iridovirus ORF 55L: GIV-55L.
was consistent with the position of GIV in iridovirus evolution. Monoclonal antibodies are a good tool for detecting and characterizing viral infection (Patil et al. 2013; Zhang et al. 2013). In recent years, mAb technology has had an important impact on aquaculture disease diagnostics (Lai et al. 2001, Ó 2015 John Wiley & Sons Ltd
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2002; Shi et al. 2003; Cote et al. 2009; Hou et al. 2011; Aamelfot et al. 2013; Patil et al. 2013; Siriwattanarat et al. 2013). In our previous study, GIV-45R(MCP), GIV-55L and GIV-97L mAbs were produced and used successfully to verify the detection and subcellular localization of target viral proteins in GIV-infected GK cells. In
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Mock infection
12 h p.i.
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Figure 6 Localization assay of GIV-64L in GIV-infected GK cells. Subcellular localization of GIV-64L was analysed using GIV64L-mAb-17 against GIV-64L by immunofluorescence assay during GIV infection of GK cells, respectively, at 0, 12 and 24 hpi. The nucleus was counter-stained by DAPI. The fluorescent signal was examined under a fluorescence microscope (Olympus IX81 Confocal Microscope, Japan).
this study, we developed and characterized three mouse mAbs (GIV-64L-mAb-1, GIV-64L-mAb17 and GIV-64L-mAb-18) that are specific to the GIV-64L protein of GIV. Further analyses demonstrated that these mAbs could serve as tools for investigating the expression and potential role of GIV-64L during GIV infection. Among the mAbs, GIV-64L-mAb-17 was the most reactive in ELISA, so we prepared GIV-64L-mAb-17 in a large quantity for subsequent studies. It proved to be an effective reagent for detecting the temporal expression and intracellular localization of GIV-64L protein during GIV infection of GK cells (Fig. 3). Generally, transcription of iridovirus genes can be classified into immediate early (IE), early and late genes according to their temporal synthesis (Williams 1996). By definition, the expression of IE genes relies solely on host proteins, which are present before viral DNA replication. The IE proteins are essential for the viral life cycle (Willis & Ó 2015 John Wiley & Sons Ltd
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Granoff 1985; Xia et al. 2010), and their 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; Holley-Guthrie et al. 1990; Williams et al. 2005; Huang et al. 2011). Expression of early genes, dependent on the preceding expression of IE genes, mainly encodes enzymes required for viral DNA synthesis and for regulating the expression of late genes. On the other hand, viral late genes are expressed after the onset of DNA replication and encode mainly structural or envelope proteins of viral particles and often are selected as vaccine candidate targets (Ebrahimi et al. 2003; Lua et al. 2005; Sanchez-Paz 2010). In our previous study, GIV-45R(MCP) and GIV55L genes were demonstrated to be late genes in GIV infection of GK cells (Hu et al. 2014; Lin et al. 2014). In the present study, the temporal expression analysis of GIV-64L was compared with that of
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GIV-45R(MCP) by RT-PCR 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. Our data showed that transcription of GIV-64L and GIV-45R(MCP) was inhibited by CHX and AraC. In addition, GIV-45R(MCP) was detected at 12 h, but GIV-64L was detected at 18 h after GIV infection (Fig. 4). The GIV-64L sequence was significantly similar to that of SGIV-93L (Singapore grouper iridovirus-93L). In a previous study, SGIV-93L was classified as a late gene by DNA microarray analysis (Chen et al. 2006) and was predicted to belong to a viral envelope protein of SGIV by proteomic analysis (Zhou et al. 2011). In the temporal translation assay of GIV64L, our data indicate that two late genes, GIV45R(MCP) and GIV-55L, were detected at 12 h, but GIV-64L was detected until 24 h after GIV infection (Fig. 4b). Overall, these data support that GIV-64L is a later gene than GIV-45R (MCP) and GIV-55L during viral replication and finally is packed into the whole virus particle. Some GIV genes localized in GIV-infected GK cells and previously characterized in our laboratory, including GIV-45R(MCP) and GIV-97L, were found to be in both the cytoplasm and nucleus, whereas GIV-55L was found only in the cytoplasm during GIV infection of GK cells. In the present study, the immunofluorescence assay with GIV64L-mAb-17 showed that GIV-64L, like GIV55L, localized only in the cytoplasm and co-localization with the virus factory during GIV infection of GK cells (Wan et al. 2010; Hu et al. 2014). In conclusion, we have cloned and identified the GIV-64L gene of GIV. GenBank searches revealed that GIV-64L homologues were present only in the members of Ranavirus. Mouse mAbs specific to GIV-64L were produced, and we have shown that these mAbs could also be valuable tools in the study of the biological function and pathological significance of GIV-64L during viral infection. Finally, GIV-64L was identified to be a very late gene and to encode a protein localized in the cytoplasm of infected cells before finally being packed into the whole virus particle. Acknowledgements This study was supported by Grant no. NSC-1022815-C-197-008-B from the National Science Council, Taiwan. Ó 2015 John Wiley & Sons Ltd
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References Aamelfot M., Weli S.C., Dale O.B., Koppang E.O. & Falk K. (2013) Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor. Journal of Anatomy 222, 547–557. Buisson M., Manet E., Trescol-Biemont M.C., Gruffat H., Durand B. & Sergeant A. (1989) The Epstein-Barr virus (EBV) early protein EB2 is a posttranscriptional activator expressed under the control of EBV transcription factors EB1 and R. Journal of Virology 63, 5276–5284. Chen L.M., Wang F., Song W. & Hew C.L. (2006) Temporal and differential gene expression of Singapore grouper iridovirus. The Journal of General Virology 87, 2907–2915. Chi S.C. (1997) The investigation of viral disease among cultured groupers in southern Taiwan. COA Fisheries Series No. 61, Reports on Fish Disease Research 18, 59–69. Chinchar V.G., Essbauer S., He J.G., Hyatt A., Miyazaki T., Seligy V. & Williams T. (2005) Family Iridoviridae. In: Virus Taxonomy: XIII Report of the International Committee on Taxonomy of Viruses (ed. by C.M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger & L.A. Ball), pp. 163–175. Elsevier, London. Chiou P.P., Chen Y.C. & Lai Y.S. (2009) Caspase-dependent induction of apoptosis in barramundi, Lates calcarifer (Bloch), muscle cells by grouper iridovirus. Journal of Fish Diseases 32, 997–1005. Chua F.H.C., Ng M.L., Ng K.L., Loo J.J. & Wee J.Y. (1994) Investigation of outbreaks of a novel disease, ‘Sleepy Grouper Disease’, affecting the brown-spotted grouper, Epinephelus tauvina Forskal. Journal of Fish Diseases 17, 417–427. Cote I., Poulos B.T., Redman R.M. & Lightner D.V. (2009) Development and characterization of a monoclonal antibody against Taura syndrome virus. Journal of Fish Diseases 32, 989–996. Ebrahimi B., Dutia B.M., Roberts K.L., Garcia-Ramirez J.J., Dickinson P., Stewart J.P., Ghazal P., Roy D.J. & Nash A.A. (2003) Transcriptome profile of murine gammaherpesvirus-68 lytic infection. Journal of General Virology 84, 99–109. He J.G., Wang S.P., Zeng K., Huang Z.J. & Chan S.M. (2000) Systemic disease caused by an iridovirus-like agent in cultured mandarinfish, Siniperca chuatsi (Basilewsky), in China. Journal of Fish Diseases 23, 219–222. Holley-Guthrie E.A., Quinlivan E.B., Mar E.C. & Kenney S. (1990) The Epstein-Barr virus (EBV) BMRF1 promoter for early antigen (EA-D) is regulated by the EBV transactivators, BRLF1 and BZLF1, in a cell-specific manner. Journal of Virology 64, 3753–3759. Hou C.L., Cao Y., Xie R.H., Wang Y.Z. & Du H.H. (2011) Characterization and diagnostic use of a monoclonal antibody for VP28 envelope protein of white spot syndrome virus. Virologica Sinica 26, 260–266. Hu S.L., Liou C.J., Cheng Y.H., Yiu J.C., Chiou P.P. & Lai Y.S. (2014) Development and characterization of two
Journal of Fish Diseases 2016, 39, 129–141
monoclonal antibodies against grouper iridovirus 55L and 97L proteins. Journal of Fish Diseases. Epub ahead of print. Huang X., Huang Y., Ouyang Z., Cai J., Yan Y. & Qin Q. (2011) Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells. The Journal of General Virology 92, 1292–1301. Lai Y.S., Murali S., Ju H.Y., Wu M.F., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2000) Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus. Journal of Fish Diseases 23, 379–388. Lai Y.S., Chiu H.C., Murali S., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2001) In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus Awoara (Temminck & Schlegel). Journal of Fish Diseases 24, 237–244. Lai Y.S., John J.A., Guo I.C., Chen S.C., Fang K. & Chang C.Y. (2002) In vitro efficiency of intra- and extracellular immunization with mouse anti-YGNNV antibody against yellow grouper nervous necrosis virus. Vaccine 20, 3221–3229. Lai Y.S., John J.A., Lin C.H., Guo I.C., Chen S.C., Fang K., Lin C.H. & Chang C.Y. (2003) Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses. Journal of Fish Diseases 26, 31–42. Lai Y.S., Chiou P.P., Chen W.J., Chen Y.C., Chen C.W., Chiu I.S., Chen S.D., Cheng Y.H. & Chang C.Y. (2008) Characterization of apoptosis induced by grouper iridovirus in two newly established cell lines from barramundi, Lates calcarifer (Bloch). Journal of Fish Diseases 31, 825–834.
tiger shrimp Penaeus monodon. Journal of Fish Diseases 36, 753–762. Pham P.H., Lai Y.S., Lee F.F., Bols N.C. & Chiou P.P. (2012) Differential viral propagation and induction of apoptosis by grouper iridovirus (GIV) in cell lines from three non-host species. Virus Research 167, 16–25. Qin Q.W., Chang S.F., Ngoh-Lim G.H., Gibson-Kueh S., Shi C. & Lam T.J. (2003) Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina. Diseases of Aquatic Organisms 53, 1–9. Reed L.J. & Muench H. (1938) A simple method of estimating fifty percent endpoints. American Journal of Hygiene 27, 493–497. Sanchez-Paz A. (2010) White spot syndrome virus: an overview on an emergent concern. Veterinary Research 41, 43. Schuh J.C.L. & Shirley I.G. (1990) Viral hematopoietic necrosis in an angelfish (Pterophyllum scalare). Journal of Zoo and Wildlife Medicine 21, 95–98. Shi C., Wei Q., Gin K.Y. & Lam T.J. (2003) Production and characterization of monoclonal antibodies to a grouper iridovirus. Journal of Virological Methods 107, 147–154. Siriwattanarat R., Longyant S., Chaivisuthangkura P., Wangman P., Vaniksampanna A. & Sithigorngul P. (2013) Improvement of immunodetection of white spot syndrome virus using a monoclonal antibody specific for heterologously expressed icp11. Archives of Virology 158, 967–979. Ting J.W., Wu M.F., Tsai C.T., Lin C.C., Guo I.C. & Chang C.Y. (2004) Identification and characterization of a novel gene of grouper iridovirus encoding a purine nucleoside phosphorylase. The Journal of General Virology 85, 2883–2892.
Langdon J.S. & Humphrey J.D. (1987) Epizootic haematopoietic necrosis, a new viral disease in redfin perch, Perca fluviatilis L., in Australia. Journal of Fish Diseases 10, 289–297.
Tsai C.T., Ting J.W., Wu M.H., Wu M.F., Guo I.C. & Chang C.Y. (2005) Complete genome sequence of the grouper iridovirus and comparison of genomic organization with those of other iridoviruses. Journal of Virology 79, 2010–2023.
Lin H.Y., Liou C.J., Cheng Y.H., Hsu H.C., Yiu J.C., Chiou P.P. & Lai Y.S. (2014) Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. Journal of Virological Methods 205, 31–37.
Wan Q.J., Gong J., Huang X.H., Huang Y.H., Zhou S., Ou-Yang Z.L., Cao J.H., Ye L.L. & Qin Q.W. (2010) Identification and characterization of a novel capsid protein encoded by Singapore grouper iridovirus ORF038L. Archives of Virology 155, 351–359.
Lua D.T., Yasuike M., Hirono I. & Aoki T. (2005) Transcription program of red sea bream iridovirus as revealed by DNA microarrays. Journal of Virology 79, 15151–15164. Murali S., Wu M.F., Guo I.C., Chen S.C., Yang H.W. & Chang C.Y. (2002) Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel). Journal of Fish Diseases 25, 91–100. Patil R., Shankar K.M., Kumar B.T., Kulkarni A., Patil P. & Moger N. (2013) Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black
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Z-Y Chen et al. Mouse mAbs against GIV-64L
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Williams T. (1996) The iridoviruses. Advances in Virus Research 46, 345–412. Williams T., Barbosa-Solomieu V. & Chinchar V.G. (2005) A decade of advances in iridovirus research. Advances in Virus Research 65, 173–248. Willis D.B. & Granoff A. (1985) trans activation of an immediate-early frog virus 3 promoter by a virion protein. Journal of Virology 56, 495–501. Wu M.S., Chen C.W., Liu Y.C., Huang H.H., Lin C.H., Tzeng C.S. & Chang C.Y. (2012) Transcriptional analysis of orange-spotted grouper reacting to experimental grouper iridovirus infection. Developmental and Comparative Immunology 37, 233–242.
Journal of Fish Diseases 2016, 39, 129–141
Ó 2015 John Wiley & Sons Ltd
Z-Y Chen et al. Mouse mAbs against GIV-64L
Xia L., Liang H., Huang Y., Ou-Yang Z. & Qin Q. (2010) Identification and characterization of Singapore grouper iridovirus (SGIV) ORF162L, an immediate-early gene involved in cell growth control and viral replication. Virus Research 147, 30–39.
Zhou S., Wan Q., Huang Y., Huang X., Cao J., Ye L., Lim T.-K., Lin Q. & Qin Q. (2011) Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088. Proteomics 11, 2236–2248.
Zhang Z., Li X., Yi W., Li S., Hu C. & Chen A. (2013) A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection. Clinical Biochemistry 46, 1105–1110.
Received: 14 July 2014 Revision received: 3 November 2014 Accepted: 4 November 2014
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