doi:10.1111/jfd.12302

Journal of Fish Diseases 2015, 38, 881–890

Identification and characterization of a late gene encoded by grouper iridovirus 2L (GIV-2L) H-Y Lin1,2, C-F Cheng1, P P Chiou3, C-J Liou4,5, J-C Yiu2 and Y-S Lai1 1 2 3 4 5

Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan Department of Horticulture, 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

Grouper iridovirus (GIV) belongs to the Ranavirus genus and is one of the most important viral pathogens in grouper, particularly at the fry and fingerling stages. In this study, we identified and characterized the GIV-2L gene, which encodes a protein of unknown function. GIV-2L is 1242 bp in length, with a predicted protein mass of 46.2 kDa. It displayed significant identity only with members of the Ranavirus and Iridovirus genera. We produced mouse monoclonal antibodies against the GIV-2L protein by immunizing mice with GIV-2L-His-tag recombinant protein. By inhibiting de novo protein and DNA synthesis in GIV-infected cells, we showed that GIV-2L was a late gene during the viral replication. Finally, immunofluorescence microscopy revealed that GIV-2L protein accumulated in both the nucleus and cytoplasm of infected cells. These results offer important insights into the pathogenesis of GIV. Keywords: grouper iridovirus, grouper iridovirus 2L, immunofluorescence, monoclonal antibodies.

Introduction

Iridoviruses are large, icosahedral, double-stranded DNA viruses with a viral particle size ranging 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 2014 John Wiley & Sons Ltd

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from 120 to 350 nm in diameter (Williams 1996; Chinchar et al. 2005). The family Iridoviridae is divided into five genera, including Ranavirus, Chloriridovirus, Lymphocystivirus, Iridovirus and Megalocytivirus. To date, iridoviruses are found to infect both invertebrates (insects) and poikilothermic vertebrates (fish, amphibians and reptiles; Eaton et al. 2007; Eaton, Ring & Brunetti 2010). Many iridoviruses can cause severe systemic diseases in their hosts, causing great economic losses in aquacultural and ecological destruction (Williams 1996). Grouper iridovirus (GIV), originally isolated from diseased grouper (Epinephelus spp.) in 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). GIV-susceptible cell lines from yellow grouper have been established (Lai et al. 2000, 2003). The complete genome of GIV has been sequenced; it consists of 139 793 bp with 49% G + C content. The genome is predicted to encode 120 open reading frames (ORFs) that range in size from 62 to 1268 amino acids (Tsai et al. 2005). In recent years, several studies have been carried out to investigate the host gene expression in response to GIV infection (Wu et al. 2012) and to characterize the apoptotic cell death caused by the virus (Lai et al. 2008; Chiou, Chen & Lai 2009; Pham et al. 2012). However, only four viral genes have been characterized during GIV infection, despite the availability of the viral genome sequence. The four viral genes are GIV-45R

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(major capsid protein; Lin et al. 2014), GIV-49L (predicted function and/or similarity to purine nucleoside phosphorylase; Ting et al. 2004), GIV55L (predicted function and/or similarity to RNase III) and GIV-97L (predicted function and/or similarity to NTPase-helicase; Hu et al. 2014). To understand the molecular mechanism of GIV pathogenesis, the expression pattern and assay tools of viral genes must be studied and developed. In this study, we cloned the GIV-2L gene from GIV and produced mouse monoclonal antibody specific to GIV-2L protein. For the first time, we have investigated its gene expression and subcellular protein localization during GIV infection in vitro.

Materials and methods

H-Y Lin et al. Monoclonal antibody against GIV-2L

phylogenetic tree was established using MEGA 6 software. Construction of pET24a-GIV-2L expression plasmids Full-length GIV-2L was cloned from GIV genomic DNA by polymerase chain reaction (PCR) with primers: GIV-2L-F/2L-R (Table 1). PCR was conducted under the following conditions: 5 min at 95 °C; 35 cycles of 45 s at 95 °C, 45 s at 56 °C, and 1 min at 72 °C; and 10 min at 72 °C. The amplified GIV-2L fragment was cloned into the pET24a expression vector. The constructs were transformed into Escherichia coli XL1-Blue, and the transformation was confirmed by restriction enzyme digestion and DNA sequencing.

Cells and viruses In this study, we used grouper kidney (GK) cells, which were previously established and characterized from the kidney mass of grouper (Lai et al. 2000). GK cells were maintained at 28 °C in L15 medium (Leibovitz) supplemented with 10% foetal bovine serum (FBS), 100 IU mL
1 penicillin and 100 lg mL
1 streptomycin. The propagation and purification of grouper iridovirus (GIV) was conducted in GK cells as described previously (Lai et al. 2000). Computer-assisted analysis GIV-2L homology analysis was performed using the BLAST network server of the National Center for Biotechnology Information. The multiple alignment of the amino acid sequence of GIV-2L with homologous sequences from 15 iridoviruses was conducted using the GeneDoc program. The

Table 1 Sequences of primers used in this study

Ó 2014 John Wiley & Sons Ltd

Name

Sequence (50 –30 )

GIV-2L-F GIV-2L-R GIV-45R (MCP)-F GIV-45R (MCP)-R GIV-55L-F GIV-55L-R b-actin-F b-actin-R

CGCGGATCCATGTCTACTTTACAGTTTATA CCGCTCGAGACTCTCCATGACATTTTCGCG CCGGAATTCATGACTTGTACAACGGGTGCT

882

CCGCTCGAGCAAGATAGGGAACCCCATGGA CGCGGATCCATGGATCAGTGGCTG CCGCTCGAGTTTAGTCTCCATCAA CCGGAATTCATGGCCATTCAACTGACACTT CCGCTCGAGAACCCCTTGGATGATAAATTC

Overproduction and purification of recombinant GIV-2L-His protein The pET24a-GIV-2L expression vector was transformed into E. coli BL21 (DE3) cells. Overexpression of GIV-2L-His recombinant protein was induced at 37 °C overnight with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG, Sigma). The cells 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. Insoluble debris was removed by centrifugation at 10 000 g for 30 min, and the supernatant was then applied directly onto a Ni-NTA Agarose affinity column (GE Healthcare Life Sciences), followed by wash with lysis buffer A containing 1, 10, 20 and 200 mM imidazole. The eluted fractions were collected and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (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) and stored at
20 °C until use. Immunization of BALB/c mice and hybridoma preparation Five female (8-week-old) BALB/c mice (National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan) were used for

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immunization. The protocols for mouse immunization and cell fusion were similar to those described by Hu et al. (2014). The selection of hybridoma clones producing monoclonal antibodies specific against GIV-2L proteins was accomplished by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence assay.

H-Y Lin et al. Monoclonal antibody against GIV-2L

PCR was completed using specific primers for GIV-2L, GIV-45R(MCP), GIV-55L and b-actin (Table 1). PCR conditions: 2 lL 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.

ELISA Ninety-six-well plates (Nunc) were coated overnight at 4 °C with 100 ng/well recombinant GIV-2L protein diluted in phosphate-buffered saline (PBS) containing 0.05% NaN3 and then blocked with 5% skim milk in PBS for 2 h at 37 °C. Plates were washed three times with PBS containing 0.05% Tween 20 (PBST), and 100 lL of mouse serum diluted in PBST or hybridoma supernatant (no dilution) was added to each well and incubated for 1 h at 37 °C. The ELISA plates were rinsed three times with PBST and patted dry on a paper towel. Next, 100 lL horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (Santa Cruz Biotechnology) diluted 1:5000 in PBST was added to each well and incubated for 1 h at 37 °C. After washing the plate three times with PBST, colour was developed by adding TMB single solution substrate (Invitrogen). After a 30min incubation in the dark, absorbances were read at 650 nm using an ELISA plate spectrophotometer reader (SpectraMax M2; Molecular Devices). Reverse transcription polymerase chain reaction (RT-PCR) To study the temporal expression pattern of the GIV-2L gene, GK cells were infected with GIV at a multiplicity of infection (MOI) of 5 and harvested at 0, 3, 6, 9, 12, 18, 24 and 30 h postinfection (hpi) in the presence or absence of cycloheximide (CHX; 50 lg mL
1; Sigma) or cytosine arabinoside (AraC; 40 lg mL
1; Sigma). Total RNA was isolated using TRIzol reagent (Invitrogen, according to the manufacturer’s protocol) and digestion with RNase-free DNase (Promega). Two micrograms of total RNA from each time point were used for first-strand cDNA synthesis by random primer following the reverse transcriptase (Roche) manufacturer’s protocol. Then, the Ó 2014 John Wiley & Sons Ltd

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Western blotting To study the temporal expression pattern of GIV2L protein, GK cells were infected with GIV (MOI = 5) and harvested at 0, 3, 6, 9, 12, 18, 24 and 30 hpi. Cells were 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); then, cell lysates were separated by electrophoresis on a 12% SDS-PAGE at 120 V for 1.5 h and transferred onto a PVDF membrane (Pall Corporation) at 400 mA for 2 h. After blocking in PBST with 5% skim milk, the membrane was incubated with GIV-2L-mAb-18 (500 lg mL
1, 1:5000), GIV-45R(MCP)-mAb21 (500 lg mL
1, 1:5000; Lin et al. 2014), GIV55L-mAb-2 (500 lg mL
1, 1:5000; Hu et al. 2014) or b-actin (3.1 mg mL
1, 1:5000; Sigma) for 2 h at 37 °C and then incubated with alkaline phosphatase-conjugated goat anti-mouse immunoglobulins (400 lg mL
1, 1:5000; Santa Cruz Biotechnology) at 37 °C for 1 h. Finally, the colour band was developed in the dark using the substrate 4-nitro-blue tetrazolium chloride/5bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Sigma). Immunofluorescence assay To analyse the location of GIV-2L protein, GK cells were seeded into a Lab-Tek chamber slideTM (Nunc) and infected with GIV (MOI = 5). At 0, 24 and 30 hpi, cells were fixed with 2% paraformaldehyde/0.1% Triton X-100 for 30 min on ice. After removal of fixative, the chamber slide was blocked with bovine serum albumin (BSA) and incubated with specific GIV-2L-mAb-18 (500 lg mL
1, 1:100) at 4 °C for 1 h. After washing three times with ice-cold PBS, the cells were incubated with fluorescein-labelled goat antimouse IgG (H + L; 500 lg mL
1, 1:50; KPL) at 4 °C for 1 h. Cellular and GIV DNA were

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labelled with 4, 6-diamidino-2-phenylindole (DAPI) for 10 min. The slides were washed three times with ice-cold PBS, mounted and observed using a confocal fluorescence microscope (Olympus IX81).

Results

Identification and sequence analysis of GIV-2L The GIV-2L (GenBank accession no. AAV91029. 1) is 1242 bp in length and encodes a putative 413-amino acid protein with a predicted molecular mass of 46.2 kDa. A 1239-bp PCR product of the GIV-2L gene was obtained by PCR from the GIV genome (Fig. 2a). Subsequent sequence analysis revealed that the PCR product contained the complete GIV-2L gene. GenBank searches revealed that GIV-2L homologues were found only in the members of the Ranavirus and Iridovirus genus. The deduced amino acid sequence of GIV-2L was aligned with the homologous sequences of 15 iridoviruses. No putative conserved domains were detected (Fig. 1a); GIV-2L demonstrated varying levels of identity with the published sequences of SGIV (99%), ADRV-1 (38%), ADRV-2 (39%), ATV (39%), ECV (39%), EHNV (39%), CGSIV (39%), CMTV (38%), RGV (38%), TFV (38%), FV3 (35%), STIV (35%), IIV6 (15%), IIV22 (13%) and WIV (13%). The abbreviations and accession numbers of 15 published iridovirus sequences are listed in Table 2. To understand the position of GIV-2L in the evolutionary history, a neighbourjoining phylogenetic tree was constructed from all 15 of the published amino acid sequences, including GIV-2L. The 15 published sequences demonstrated significant identity only to members of the Ranavirus and Iridovirus genuses (Fig. 1b). GIV-2L and other gene members in the genus Ranavirus were clustered within a monophyletic group with a 100% bootstrap. Interestingly, the GIV-2L phylogenetic tree was consistent with the relationship of two genera within the family Iridoviridae. Expression and purification of recombinant GIV-2L-His protein The recombinant GIV-2L-His protein was expressed successfully in E. coli BL21 (DE3) cells. A band with expected molecular weight of Ó 2014 John Wiley & Sons Ltd

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H-Y Lin et al. Monoclonal antibody against GIV-2L

approximate 48.6 kDa (435 amino acids) was identified in the cell lysate after 24 h of induction with 1 mM IPTG, and the recombinant GIV-2LHis protein was further purified (Fig. 2b) to immunize mice for monoclonal antibody production. Production and characterization of GIV-2L monoclonal antibodies Two stable, positive hybridoma lines (GIV-2LmAb-2 and GIV-2L-mAb-18) producing antibodies against GIV-2L were identified by ELISA and Western blot. As shown in Fig. 2c, the specificity of GIV-2L-mAb-18 was demonstrated by Western blot analysis. In addition, both GIV-2L-mAb-2 and GIV-2L-mAb-18 belong to the IgG1 isotype (data not shown). Transcriptional and translational expression patterns of GIV-2L in GIV-infected GK cells The transcriptional and translational expression patterns of GIV-2L in GIV-infected GK cells were analysed first by RT-PCR and then by Western blotting with GIV-2L-mAb-18 at 3, 6, 9, 12, 18, 24 and 36 hpi. GIV-45R(MCP) and GIV-55L transcripts were detectable by PCR at as early as 12 hpi, and GIV-2L transcript was detectable at 24 hpi (Fig. 3a). To further verify the temporal expression of GIV-2L protein,

Table 2 GenBank accession numbers of GIV-2L homologs used in this study Name of species (Abbreviation) Groper iridovirus (GIV) Singapore Groper Iridovirus (SGIV) Andrias davidianus ranavirus-1 (ADRV-1) Andrias davidianus ranavirus-2 (ADRV-2) Ambystoma tigrinum virus (ATV) Chinese giant salamander iridovirus (CGSIV) Common midwife toad ranavirus (CMTV) European catfish virus (ECV) Epizootic haematopoietic necrosis virus (EHNV) Rana grylio iridovirus (RGV) Tiger frog virus (TFV) Frog virus 3 (FV3) Soft-shelled turtle iridovirus (STIV) Invertebrate iridescent virus 6 (IIV-6) Invertebrate iridovirus 22 (IIV-22) Wiseana iridescent virus (WIV) (IIV-9)

Accession number AAV91029.1 YP164111.1 AGV20535.1 AHA42272.1 YP003774.1 AHA80848.1 AFA44908.1 YP006347594.1 AC025193.1 AFG73046.1 ABB92272.1 YP031581.1 ACF42224.1 NP149692.1 YP008357435.1 YP004732868.1

H-Y Lin et al. Monoclonal antibody against GIV-2L

Journal of Fish Diseases 2015, 38, 881–890

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: : : : : : : : : : : : : : : :

(b)

Figure 1 Sequence alignment and phylogenetic analysis of GIV-2L. (a) Multiple amino acid sequence alignment of GIV-2L with related gene sequences from 15 other iridoviruses. The identical residues in all sequences are marked by a pound symbol (*). (b) Phylogenetic analysis of GIV-2L and related proteins in the family Iridoviridae. The phylogenetic tree was constructed using neighbour-joining method. Branch lengths are proportional to the evolutionary distance between the taxa.

GIV-2L expression was assayed in GIV-infected cells in the presence of CHX (an inhibitor of de novo protein synthesis) or AraC (an inhibitor Ó 2014 John Wiley & Sons Ltd

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of DNA synthesis). As shown in Fig. 3b, transcription of GIV-2L, GIV-45R(MCP) and GIV55L was inhibited by CHX and AraC. This

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Journal of Fish Diseases 2015, 38, 881–890

(a)

(b)

(c)

Figure 2 GIV-2L gene isolation, recombinant protein expression and purification, and mouse monoclonal antibody production. (a) PCR amplification of the GIV-2L 1239-bp fragment from GIV genomic DNA. (b) The expressed and purified recombinant GIV2L-His protein was separated using SDS-PAGE. The gel was stained with Coomassie blue after electrophoresis. M, prestained protein marker; Lane 1, non-IPTG-induced cell lysate; Lane 2, 1 mM IPTG-induced cell lysate; Lane 3, purified GIV-2L-His protein. (c) Production and characterization of mouse monoclonal antibody against recombinant GIV-2L-His protein. M, prestained protein marker; Lane 1, non-IPTG-induced cell lysate; Lane 2, 1 mM IPTG-induced cell lysate; and Lane 3, purified GIV-2L-His protein.

(a)

0

3

6

9

12

18

24

30

+AraC (b) + CHX 12 24 30 hpi 12 24 30 hpi

hpi GIV-2L

GIV-2L

GIV-45R(MCP)

GIV-45R(MCP)

GIV-55L

GIV-55L

β-Actin Figure 3 Temporal expression pattern of GIV-2L, GIV-45R(MCP) and GIV-55L transcripts in GIV-infected GK cells. (a) GK cells were infected with GIV (multiplicity of infection, MOI = 5), and the transcripts were assayed by RT-PCR at 0, 3, 6, 9, 12, 18, 24 and 30 hpi. (b) GK cells were infected with GIV (MOI = 5) in the presence of CHX or AraC, and the transcripts were assayed by RT-PCR at 12, 24 and 30 hpi.

finding supports the notion that GIV-2L is expressed as a viral late gene in GIV-infected GK cells. GIV-55L and GIV-45R(MCP) proteins were detected at 9 and 12 hpi, respectively; in contrast, GIV-2L was not detected until 24 h after infection (Fig. 4b). This result demonstrates that the GIV-2L-mAb-18 monoclonal antibody can be used as an effective detection tool in Western blotting. Based on the RT-PCR and Western blot analyses, GIV-2L might be a viral late gene. Ó 2014 John Wiley & Sons Ltd

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Intracellular localization of GIV-2L in GIVinfected GK cells The monoclonal antibody GIV-2L-mAb-18 was used in an immunofluorescence assay to examine the intracellular localization of GIV-2L in GIVinfected GK cells. GIV-2L-mAb-18 detected GIV2L protein in GIV-infected GK cells at 24 and 30 hpi (Fig. 5). The green fluorescent signal from GIV-2L was distributed in both the nucleus and the cytoplasm of GIV-infected GK cells (Fig. 5). This result demonstrates that in addition to its

H-Y Lin et al. Monoclonal antibody against GIV-2L

Journal of Fish Diseases 2015, 38, 881–890

(a)

kDa 75 63

M

0

3

6

9 12 18 24 30 hpi

(b)

0

3

6

9

12 18

24

30 hpi

GIV-2L

48 35 25

GIV-45R(MCP)

GIV-55L

17 11

β-Actin

Figure 4 Temporal expression pattern of GIV-2L, GIV-45R(MCP) and GIV-55L proteins in GIV-infected GK cells. (a) GK cells were infected with GIV at an MOI of 5, respectively, at 0, 3, 6, 9, 12, 18, 24 and 30 hpi, and cell lysates were separated by SDSPAGE. Gel was stained with Coomassie blue after electrophoresis. M: prestained protein marker. (b) GK cells were infected with GIV at an MOI of 5, and the translations of GIV-2L, GIV-45R(MCP) and GIV-55L were measured by Western blotting analysis, respectively, at 0, 3, 6, 9, 12, 18, 24 and 30 hpi.

Figure 5 Localization of GIV-2L in GIV-infected GK cells. Subcellular localization of GIV-2L was analysed using a specific mouse monoclonal antibody against GIV-2L and an immunofluorescence assay at 0, 24 and 30 hpi. The nucleus was counter-stained using DAPI. The fluorescent signal was examined under a fluorescence microscope (Olympus IX81 Confocal Microscope). Ó 2014 John Wiley & Sons Ltd

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Journal of Fish Diseases 2015, 38, 881–890

use as an effective Western blot detection tool, GIV-2L-mAb-18 is also appropriate for use in immunofluorescence assays. Discussion

Grouper (Epinephelus spp.) is an economically important fish species worldwide. GIV is one of the most important viral pathogens and has resulted in significant economic losses in the grouper aquaculture industry (Chi 1997; Lai et al. 2000, 2001). Although the viral genome sequence is now known, the molecular mechanisms underlying the pathogenicity of iridovirus are still not well understood, partly due to insufficient tool for viral protein detection. The GIV genome sequence, host cellular gene expression and apoptotic cell death characteristics have been investigated in recent years (Lai et al. 2008; Chiou et al. 2009; Pham et al. 2012; Wu et al. 2012). However, only four functional genes, encoding four proteins, have been identified and characterized in infected host cells: GIV-45R(MCP), GIV-49L, GIV-55L, and GIV-97L (Ting et al. 2004; Hu et al. 2014; Lin et al. 2014). In the present study, we have developed an immunological reagent to detect a late gene 2L of GIV. The evolutionary relationship of GIV-2L with other homologues was consistent with the position of GIV in iridovirus evolution. Monoclonal antibodies are a good tool for the detection and characterization of viruses (Patil et al. 2013; Zhang et al. 2013). Monoclonal antibody technology has made important contribution to aquaculture disease diagnosis in recent years (Lai et al. 2001, 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 this study, we developed and characterized two mouse monoclonal antibodies (GIV-2L-mAb-2 and GIV2L-mAb-18) that are specific to the GIV-2L protein. Furthermore, we characterized these antibodies as of IgG1 isotype (data not showed). Further analysis demonstrated that these two monoclonal antibodies were able to serve as potent tools to investigate the expression and potential role of GIV-2L during GIV infection. The transcription of iridovirus genes can generally be classified as immediate early (IE), early (E) or late (L) according to the timing of their synthesis (Williams 1996). By definition, the expression of IE genes relies solely on host proteins, which do not require viral DNA replication. The IE Ó 2014 John Wiley & Sons Ltd

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proteins are often essential for the viral life cycle (Willis & Granoff 1985; Xia et al. 2010); their function might be involved in activating the expression of viral early and late genes, altering the functions of host genes and eliminating the host immune defence (Buisson et al. 1989; Holley-Guthrie et al. 1990; Williams, Barbosa-Solomieu & Chinchar 2005; Huang et al. 2011). E gene expression, which is dependent on the preceding expression of IE genes, mainly encodes enzymes required for viral DNA synthesis and for the regulation of L genes’ expression. Viral L genes are expressed after the onset of DNA replication; they mainly encode structural or envelope proteins of viral particles (Ebrahimi et al. 2003; Lua et al. 2005; Sanchez-Paz 2010). In a previous study, we used GIV infection of GK cells to demonstrate that GIV-45R(MCP) and GIV-55L genes belong to the L gene group (Hu et al. 2014; Lin et al. 2014). In the present study, we used RT-PCR to compare the temporal expression of GIV-2L, GIV-45R(MCP) and GIV-55L in the presence of inhibitors against DNA or protein synthesis. Our data showed that transcription of all three genes (GIV-2L, GIV-45R(MCP) and GIV-55L) was inhibited by CHX and AraC. In addition, GIV-55L and GIV-45R(MCP) were detected at 9 and 12 h after GIV infection, respectively, while GIV-2L was detected at 24 hpi (Fig. 4b). Overall, these data support that GIV-2L is a late gene during viral replication and also demonstrates the validity of the GIV-2L-mAb-18 as a useful analytical tool. GIV-2L sequence shows significant identity with the 16L protein of SGIV (Singapore grouper iridovirus). In a previous study, SGIV-16L was classified as an early gene by DNA microarray analysis (Chen et al. 2006) and further shown to be a viral envelope protein with a predicted transmembrane domain structure (Song et al. 2006; Zhou et al. 2011). Being an envelope protein, SGIV 16L has been tested as a candidate antigen to generate anti-SGIV vaccine; unfortunately, the viral protein failed to elicit significant protection against the SGIV infection (Ou-Yang et al. 2012). In our previous study, we produced monoclonal antibodies against GIV-45R, GIV-55L and GIV97L and used them to identify the subcellular localization of target viral proteins. Both GIV45R and GIV-97L were localized in the cytoplasm and the nucleus, while GIV-55L was observed in the cytoplasm only. In the present study, an

Journal of Fish Diseases 2015, 38, 881–890

immunofluorescence assay with GIV-2L-mAb-18 demonstrated that GIV-2L is located in both the cytoplasm and the nucleus. In conclusion, we have cloned and identified the 2L gene of GIV. We have produced mouse monoclonal antibodies specific to the GIV-2L protein, and these monoclonal antibodies may be valuable tools for future studies of the biological function and pathological significance of GIV-2L during viral infection. Finally, we confirmed that GIV-2L is a late gene and encodes a protein that is located in both the cytoplasm and the nucleus of infected cells. Acknowledgements This study was supported by Grant no. NSC-1012815-C-197-011-B from the National Science Council, Taiwan.

(Bloch), muscle cells by grouper iridovirus. Journal of Fish Diseases 32, 997–1005. 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. Eaton H.E., Metcalf J., Penny E., Tcherepanov V., Upton C. & Brunetti C.R. (2007) Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes. Virology Journal 4, 11. Eaton H.E., Ring B.A. & Brunetti C.R. (2010) The genomic diversity and phylogenetic relationship in the family iridoviridae. Viruses 2, 1458–1475. 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.

Publication History

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.

Received: 6 June 2014 Revision received: 29 June 2014 Accepted: 21 July 2014

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.

This paper was edited and accepted under the Editorship of Professor Ron Roberts.

Hu S.L., Liou C.J., Cheng Y.H., Yiu J.C., Chiou P.P. & Lai Y.S. (2014) Development and characterization of two monoclonal antibodies against grouper iridovirus 55L and 97L proteins. Journal of Fish Diseases, Epub ahead of print.

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