G Model

ARTICLE IN PRESS

VIRMET 12506 1–7

Journal of Virological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet

Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein

1

2

3 4 5 6 7 8 9

Q1

Hong-Yi Lin a,d , Chian-Jiun Liou b,c , Yeong-Hsiang Cheng d , Hui-Chen Hsu d , Jinn-Chin Yiu a , Pinwen Peter Chiou e , Yu-Shen Lai d,∗ a

Department of Horticulture, National Ilan University, Yilan, Taiwan Department of Nursing, Chang Gung University of Science and Technology, Taoyuan, Taiwan c Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan d Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan e Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan b

10

a b s t r a c t

23 11 12 13 14 15 16

Article history: Received 9 January 2014 Received in revised form 10 April 2014 Accepted 16 April 2014 Available online xxx

17

22

Keywords: Major capsid protein Grouper Iridovirus Monoclonal antibody

24

1. Introduction

18 19 20 21

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

The major capsid protein (MCP) is a main structural protein of iridoviruses, and is used as a marker for the identification, differentiation and classification of ranaviruses. In the present study, six monoclonal antibodies (mAbs) against recombinant MCP of grouper iridovirus (GIV) were produced and characterized. All of the six mAbs were of IgG1 isotype. Among the mAbs, GIV-MCP-mAb-21 showed the highest reactivity in ELISA and was used to further characterize the expression of GIV-MCP during viral replication. RT-PCR and Western blot analyses revealed that GIV-MCP is a late gene during GIV infection. By immunofluorescence assay, the presence of GIV-MCP was observed in not only the cytoplasm but also the nucleus of GIV-infected cells, a surprising finding that might indicate additional role of GIV-MCP. In conclusion, the newly established GIV-MCP-mAbs are a valuable tool for GIV diagnostic and future studies on GIV pathogenesis. © 2014 Elsevier B.V. All rights reserved.

Iridoviruses are large icosahedral viruses containing a single molecule of linear double-stranded DNA packed in a viral particle with diameter ranging from 120 to 350 nm (Williams, 1996). The Iridoviridae family includes five genera—Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and Megalocytivirus—whose members can infect only cold-blooded vertebrates and invertebrates (Chinchar et al., 2005; Williams et al., 2005). Iridoviruses can cause severe diseases in various aquatic animals, especially in East and Southeast Asian marine-cultured fish species. Infectious disease is a major constraint in hatchery production of grouper, one of the most economically important cultured marine fish in many Asian countries (Chi, 1997; Lai et al., 2001a,b). Iridoviruses are one group of the most important viral pathogens in grouper, particularly at the fry and fingerling stages (Lai et al., 2000, 2003). Grouper iridovirus (GIV), a member of the Ranavirus genus

∗ Corresponding author at: Department of Biotechnology and Animal Science, National Ilan University 1, Sec. 1, Shen-Lung Road, Yilan 26047, Taiwan. Tel.: +886 3 9357400 7624. E-mail address: [email protected] (Y.-S. Lai).

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). The early diagnosis and prevention of GIV infection is critical to sustaining the grouper aquaculture industry. Several methods have been developed for diagnostic of iridoviral diseases, including clinical signs, microscopic pathological signs and nucleic acid diagnostic methods. Fish infected with iridovirus might show darken body color, pale gills, enlarged spleen, and often become lethargic with low appetite (Langdon and Humphrey, 1987; Schuh and Shirley, 1990; Chua et al., 1994; He et al., 2000; Lai et al., 2000). In the later stages of the disease, fish might also exhibited rapid opercula movements, typified by dashing to the surface for air. Fish might also show deep ulceration in muscular tissue and red boils on the body surface due to secondary infection. Microscopic pathological signs might include enlargement of cells and necrosis of renal and splenic hematopoietic tissues (Qin et al., 2003). Both the clinical sign and microscopic sign serve as a primary diagnostic of the disease. PCR-based nucleic acid diagnostic methods, including a loop-mediated isothermal amplification (LAMP) method (Sung et al., 2010), have been developed to detect the presence of viral nucleic acid sequences in the infected fish. The PCR-based methods are sensitive and accurate, but require

http://dx.doi.org/10.1016/j.jviromet.2014.04.013 0166-0934/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

G Model VIRMET 12506 1–7

ARTICLE IN PRESS

2

H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

92

expertise in handling, thus not suitable for operation in nonlaboratory settings. On the other hand, immunological assays require less technical expertise and specific equipment. However, proper GIV-specific antibodies for immunological assays are not available yet. Monoclonal antibodies (mAbs) are efficient tools for detection and characterization of pathogens. For example, specific mAb against the nucleocapsid protein of Schmallenberg virus (SBV) can be used to detect SBV infection effectively (Zhang et al., 2013a). In the case of Singapore grouper iridovirus (SGIV), a ranavirus, mAbs have been developed to characterize the subcellular localization of viral proteins in grouper cells (Shi et al., 2003). In addition to the detection and characterization of pathogens, mAbs are applied broadly in disease therapy and immunotherapy against cancers. For examples, a mAbs against HCV host entry factor CD81 blocks HCV spreading and dissemination efficiently (Fofana et al., 2013); the IP-10 mAb against influenza A (H1N1) improves the survival rate and ameliorates the acute lung injury in the infected mice (Wang et al., 2013). An example of successful application of mAbs in cancer immunotherapy is the anti-p185HER2 murine monoclonal antibody (generic name: Trastuzumab). Trastuzumab can inhibit the growth of human breast tumor in nude mice and is regarded currently as a first-line for metastatic breast cancers that overexpress Her-2 (Toi et al., 2004). Major capsid protein (MCP) is a predominant structural component of iridovirus particles, and has been used as a marker for ranavirus differentiation and classification (Tidona et al., 1998). In this study, mAbs against GIV-MCP were developed and characterized. These specific mAbs are a valuable tool for GIV diagnostic and future studies on GIV pathogenesis.

93

2. Materials and methods

94

2.1. Grouper kidney (GK) cell lines and GIV preparation

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91

98

GK cells were grown at 28 ◦ C in Leibovitz’s L15 medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 ␮g/mL streptomycin. GIV propagation and purification were performed in GK cells as described previously (Lai et al., 2000).

99

2.2. Cloning and plasmid construction of the GIV-MCP gene

95 96 97

NJ, USA) and washed sequentially with lysis buffer A containing 1, 5, 10, 20, and 200 mM imidazole (Sigma, St. Louis, MO, USA). The eluent was collected in 3-mL fractions, which were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing protein of the expected molecular weight were pooled and dialyzed with S100 buffer (25 mM HEPES; 20% glycerol; 100 mM KCl; 0.2 mM EDTA; and 1 mM DTT, pH 7.9). After dialysis, the purified recombinant GIVMCP-His concentration was measured using the Bradford method (Bradford, 1976). The purified protein was used as the immunogen to generate mAbs. 2.4. Immunization of mice and hybridoma preparation Pathogen-free 12-week-old BALB/c mice were purchased from the National Laboratory Animal Breeding and Research Center, National Science Council, Taiwan. Animal experiments were performed in accordance with the guidelines of the National Laboratory Animal Center on Institutional Animal Care and Use Committee (IACU). Each of four mice was injected intraperitoneally with 100 ␮g of GIV-MCP-His protein mixed with an equal volume of Freund’s complete adjuvant (Sigma, St. Louis, MO, USA). Two weeks later, the mice were given a booster injection of the same dosage but prepared in Freund’s incomplete adjuvant (Sigma, St. Louis, MO, USA). Two more injections were administered at two-week intervals. Three days after the last injection, mouse serum was collected to test the production of polyclonal antibodies against the GIV-MCPHis protein. Additionally, spleen cells were removed and fused with mouse myeloma cells (NS-1) in the presence of 50% polyethylene glycol (MW 1500). After fusion, hybrid cells were distributed in 96-well plates and cultured in RPMI1640 medium supplemented with 10% FBS and 1× hypoxanthine–aminopterin–thymidine (HAT; Sigma, St. Louis, MO, USA) for 7–10 days. ELISA was performed to screen the hybridoma culture media for the presence of specific antibodies against GIV-MCP-His protein. Hybridoma cells from wells with a positive signal were further subcloned by limiting dilution, and screened again by ELISA. 2.5. ELISA

110

The complete GIV-MCP (GIV45R) sequence was amplified from GIV genomic DNA by PCR using oligonucleotide primers containing restriction enzyme cleavage sites: forward5 -CCGGAATTCATGACTTGTACAACGGGTGCT-3 (EcoRI site underlined), and reverse-5 -CCGCTCGAGCAAGATAGGGAACCCCATGGA3 (XhoI site underlined). PCR conditions were as follows: 10 min at 95 ◦ C; 35 cycles of 1 min at 95 ◦ C, 1 min at 56 ◦ C, and 1 min at 72 ◦ C; and a final extension for 10 min at 72 ◦ C. The amplified GIV-MCP gene was ligated into the prokaryotic expression vector pET23a using the YB rapid ligation kit (Yeastern Biotech; Taipei, Taiwan) according to the manufacturer’s protocol.

111

2.3. Production and purification of recombinant GIV-MCP-His

100 101 102 103 104 105 106 107 108 109

112 113 114 115 116 117 118 119 120 121

The pET23a-GIV-MCP plasmid was transformed into Escherichia coli BL21 (DE3), and the expression of GIV-MCP-His fusion proteins was induced with 1 mM isopropyl ␤-d-1thiogalactopyranoside (IPTG) (Sigma, St. Louis, MO, USA) for 24 h at 37 ◦ C. The cells were lysed overnight at 4 ◦ C with lysis buffer A (6 M guanidine hydrochloride; 0.1 M NaH2 PO4 ; and 0.01 M Tris, pH 8.0), using 5 mL buffer A per 1 g cell pellet. The insoluble debris was removed by centrifugation at 10,000 × g for 30 min. The supernatant was applied directly onto a Ni-NTA agarose affinity chromatography column (GE Healthcare Life Sciences, Piscataway,

In ELISA plates (Nunc, Thermo Scientific; Roskilde, Denmark), each well was coated with 100 ␮L of GIV-MCP-His protein suspension (1 ng/␮L) diluted in phosphate-buffered saline (PBS; pH 7.2) containing 0.05% NaN3 , and incubated overnight at 4 ◦ C. After discarding non-absorbed antigen, the plates were washed three times with PBS containing 0.05% (v/v) Tween20 (PBST), then blocked with 5% skim milk in PBS for 2 h at 37 ◦ C. Next, the plates were washed three times with PBST, and 100 ␮L of hybridoma supernatant diluted in PBST was added to each well. After 2 h incubation at 37 ◦ C, the plates were rinsed three times with PBST, and 100 ␮L of 1:5000-diluted horseradish peroxidase-conjugated goat antimouse immunoglobulin (Santa Cruz Biotechnology; Santa Cruz, CA, USA) was added to each well. The plates were incubated for 1 h at 37 ◦ C. The plates were washed once more with PBST, and then the enzyme activity was determined by adding 100 ␮L of the TMB single solution substrate (Invitrogen; Paisley, Scotland, UK). After incubation at room temperature for 30 min in the dark, the optical density (OD) of each well was determined at 650 nm using an ELISA SpectraMax M2 plate reader (Molecular Devices; Sunnyvale, CA, USA). 2.6. Mouse mAb isotype determination and purification The six mAbs against GIV-MCP protein were isotyped using a mouse mAb isotyping kit (Sigma; St. Louis, MO, USA) according

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

122 123 124 125 126 127 128 129 130 131 132

133

134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157

158

159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178

179

180 181

G Model VIRMET 12506 1–7

ARTICLE IN PRESS H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

182 183 184 185 186 187 188

189 190

to the manual. Following IACU guidelines, ascitic fluids were generated by intraperitoneal injection of each 20-week-old BALB/c mice with 200 ␮L pristine and then with 5 × 106 hybridoma cells. The mAbs were purified from the ascitic fluids using saturated ammonium sulfate solution and NAbTM protein G spin kit (Thermo Scientific; Rockford, IL, USA) according to the manufacturer’s protocol. 2.7. Total RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)

209

GK cells were infected with GIV at a multiplicity of infection (MOI) of 5, and were harvested at 0, 4, 8, 12, 24, 30, and 36 h post-infection (hpi) in the presence or absence of cycloheximide (CHX; 50 ␮g/mL; Sigma; St. Louis, MO, USA) or arabinoside (AraC; 40 ␮g/mL; Sigma; St. Louis, MO, USA). Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Paisley, Scotland, UK) according to the manufacturer’s protocol. After treatment with RNase-free DNase (New England Biolabs; Beverly, MA), 2 ␮g of total RNA was reverse-transcribed into first-strand cDNA using random primers and reverse transcriptase (Roche; Penzberg, Germany) following the manufacturer’s protocol. The PCR was carried out in a volume of 50 ␮L, containing 2 ␮L cDNA, 0.5 ␮M forward primer, 0.5 ␮M reverse primer, 2.5 ␮M dNTP, 1× PCR buffer, and 2.5 U Taq DNA polymerase (Viogene; Taipei, Taiwan). PCR conditions were as follows: 10 min at 95 ◦ C; 35 cycles of 1 min at 95 ◦ C, 1 min at 56 ◦ C, and 1 min at 72 ◦ C; and a final extension of 10 min at 72 ◦ C. ␤-Actin mRNA was used as an internal control. After amplification, the PCR products were electrophoresed in a 1.2% agarose-TAE buffer gel and stained with ethidium bromide.

210

2.8. Western blot analysis

191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

231

The six GIV-MCP mAbs were analyzed by Western blot. The purified GIV particles (PGIVP) and GIV-MCP-His fusion proteins were separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Corporation; East Hills, NY, USA). After blocking with 5% skim milk overnight at 4 ◦ C, the membrane was incubated with the non-diluted culture medium of the six hybridoma clones for 2 h at 37 ◦ C. The GIV-MCP protein expression pattern was also determined in GK cells during GIV infection with an MOI of 5. GIV-infected GK cells were lysed in 2× SDS sample buffer at 4, 8, 12, 24, 30, and 36 hpi. Cell lysates were subjected to 12% SDS-PAGE and transferred onto a PVDF membrane. The membrane was washed in PBST and blocked with 5% skim milk overnight at 4 ◦ C. After washing, the membrane was reacted with GIV-MCP mAb-21 or ␤-actin antibody (Sigma, St. Louis, MO, USA) for 2 h at 37 ◦ C. The membrane was then washed, and incubated for 1 h at 37 ◦ C with a secondary antibody (1:5000) (alkaline phosphatase-conjugated goat anti-mouse immunoglobulins; Santa Cruz Biotechnology; Santa Cruz, CA, USA). The specific signal bands were visualized with 5-bromo-4-chloro3-indolyl phosphate (BCIP) and 4-nitro-blue tetrazolium chloride (NBT) (Sigma; St. Louis, MO, USA).

232

2.9. Localization of GIV-MCP in GIV-infected GK cells

211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230

3

with fluorescein-isothiocyanate (FITC) was incubated at 4 ◦ C for 1 h. Cellular DNA was stained with 4 ,6-diamidino-2-phenylindole (DAPI), followed by fluorescence microscopy (Axio Observer; Zeiss, Jena, Germany).

234 235 236 237 238 239 240 241

GK cells grown on a four-well Lab-Tek chamber slideTM (Nalge Nunc; Napersville, IL, USA) were infected with GIV at an MOI of 5. At 0, 24, and 36 hpi, cells were washed with PBS and fixed in 2% para-formaldehyde/0.1% Triton X-100 for 30 min on ice. After removal of fixative, the chamber slides were blocked by incubation with bovine serum albumin (BSA) for 1 h, followed by incubation with GIV-MCP mAb-21 (0.5 ␮g) at 4 ◦ C for 1 h. After washing with ice-cold PBS, the secondary antibody (1:50) (Polyclonal Rabbit AntiMouse Immunoglobulins; Dako, Glostrup, Denmark) conjugated

243 244 245

3. Results

246

3.1. Sequence analysis of GIV-MCP

247

The GIV-MCP (GIV45R; accession no. AAV91066) sequence comprised 1392 bp, encoding a putative 463-amino acid protein with a predicted molecular mass of 50.59 kDa (Fig. 1). Its deduced amino acid sequence was 99%, 72%, 72%, 73%, 72%, and 71% identical to the homologs of Singapore grouper iridovirus (SGIV; accession no. AAS18087), frog virus 3 (FV3; accession no. ACP19256), tiger frog virus (TFV; accession no. AAK55105), soft-shelled turtle iridovirus (STIV; accession no. ABC59813), Ambystoma tigrinum stebbinsi virus (ATV; accession no. AAP33191), and epizootic hematopoietic necrosis virus (EHNV; accession no. AAO32315), respectively (Fig. 2). 3.2. Expression and purification of recombinant GIV-MCP The pET23a-GIV-MCP plasmid was constructed to express the 53.24-kDa recombinant GIV-MCP-His protein. Optimal GIV-MCPHis expression was obtained by incubation with 1 mM IPTG for 24 h at 37 ◦ C (Fig. 3A). The recombinant GIV-MCP-His protein was purified successfully (Fig. 3A), and was later identified by Western blot with anti-GIV-MCP-His polyclonal serum (Fig. 3B). 3.3. Production and characterization of mouse mAbs against GIV-MCP Mice were immunized with GIV-MCP-His protein to elicit an antibody response and to generate hybridoma cells. Subsequently, the hybridoma clones were screened by ELISA against GIV-MCP-His. Out of 73 clones, six were found to be positive against GIV-MCP-His protein: GIV-MCP-mAb-17, OD650 = 0.66; GIV-MCP-mAb-21, OD650 = 0.89; GIV-MCP-mAb-27, OD650 = 0.69; GIV-MCP-mAb-53, OD650 = 0.72; GIV-MCP-mAb-61, OD650 = 0.59; and GIV-MCP-mAb-63, OD650 = 0.41. ELISA-positive mAb supernatants were further verified by Western blot using either the corresponding recombinant GIV-MCP-His protein or purified GIV particles (PGIVP). All six ELISA-positive mAbs recognized specifically the GIV-MCP-His protein and PGIVP (Fig. 4A). Interestingly, mAbs 27 and 53 appeared to yield a stronger band than mAb21 did, while mAb21 exhibited the highest reactivity in the ELISA. Further isotyping showed that all obtained GIV-MCP-mAbs were of IgG1 isotype (Fig. 4B). Hence, the data indicate that these mAbs recognize different epitopes of GIV-MCP. Nevertheless, as ELISA is a more sensitive method than Western blot in general and is more suitable than Western blot for analysis of samples in large quantity, we proceeded to further characterize GIV-MCP-mAb-21 and verify the feasibility of the mAbs for other immunological assays to detect the presence of GIV-MCP. 3.4. Purification and dilution limit of mouse GIV-MCP-mAb-21

233

242

To prepare GIV-MCP-mAb-21 in large quantity for subsequent studies, GIV-MCP-mAb-21 hybridoma cells were injected into mice to produce ascites, from which the mAbs were prepared successfully after the treatments with saturated ammonium sulfate solution and protein G agarose (Fig. 5A). We then analyzed the detection sensitivity of the prepared mAB-21 in Western blot analysis. In the assay, the antigen concentration was 1 ␮g PGIVP/per lane and GIV-MCP-mAb-21 was applied in a serial dilution (1 ␮g,

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

248 249 250 251 252 253 254 255 256 257 258

259

260 261 262 263 264 265

266 267

268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289

290

291 292 293 294 295 296 297 298

G Model VIRMET 12506 1–7

ARTICLE IN PRESS

4

H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

Fig. 1. GIV-MCP gene isolation. (A) PCR amplification of GIV-MCP fragment from GIN genomic DNA. (B) Nucleotide identification and amino acid sequence alignment of GIV-MCP.

299 300 301 302 303

304

305 306 307

0.5 ␮g, 0.1 ␮g, and 0.05 ␮g). Protein signals were visualized with a goat anti-mouse secondary antibody conjugated to AP (1:5000) and BCIP/NBT substrate. As shown in Fig. 5B, GIV-MCP-mAb-21 could detect GIV-MCP of PGIVP with an approximate detection limit of 0.05 ␮g under non-reducing conditions. 3.5. Expression pattern of GIV-MCP in the course of GIV infection Experiments were also performed to verify the feasibility of GIV-MCP-mAb-21 in Western blot assay to detect the temporal expression of GIV-MCP in GIV-infected cells. The expression of

GIV-MCP was first analyzed by RT-PCR, and then by Western blot with the GIV-MCP-mAb-21 at 4, 8, 12, 24, 30, and 36 hpi (Fig. 6). RT-PCR analysis detected the GIV-MCP transcript at 12 hpi (Fig. 6A), but the protein was not detected until 24 hpi (Fig. 6B). This result demonstrated that the GIV-MCP-mAb-21 can be used as an effective reagent in Western blot. Furthermore, the RT-PCR and Western blot results indicate that GIV-MCP might be a late gene. The expression of GIV-MCP gene was further assayed in GIV-infected cells in the presence of CHX or AraC. CHX is an inhibitor of de novo protein synthesis, and AraC is a pyrimidine anti-metabolite that can block DNA synthesis. As shown in Fig. 6A, GIV-MCP transcription was

Fig. 2. Multiple amino acid sequence alignment of the MCP gene of GIV with related gene sequences of six other iridoviruses of the Ranavirus genus. The gray boxes indicate identical sequences.

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

308 309 310 311 312 313 314 315 316 317 318

G Model VIRMET 12506 1–7

ARTICLE IN PRESS H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

Fig. 3. Expression and purification of recombinant GIV-MCP protein, and production of anti-GIV-MCP mouse polyclonal antibody. (A) SDS-PAGE analysis of expression and purification of recombinant GIV-MCP protein in E. coli. Gel was stained with coomassie blue after electrophoresis. M, pre-stained protein marker; lane 1, non-IPTG-induced cell lysate; lane 2, IPTG-induced cell lysate; lane 3, purified GIV-MCP-His protein. (B) Production and characterization of mouse polyclonal antibody against recombinant GIV-MCP protein. M, pre-stained protein marker; lane 1, non-IPTG-induced cells lysate; lane 2, IPTG-induced cells lysate; lane 3, purified GIV-MCP-His protein.

320

inhibited by CHX and AraC, supporting the notion that GIV-MCP is a late gene.

321

3.6. Intracellular localization of GIV-MCP

319

331

Lastly, we verified if GIV-MCP-mAb-21 could be used to detect the intracellular localization of GIV-MCP by immunofluorescence microscopy. As shown in Fig. 7, GIV-MCP-mAb-21 recognized the presence of GIV-MCP protein in the GIV-infected GK cells at 24 and 36 hpi. Interestingly, the green fluorescence signals of GIVMCP signals were distributed in both the nucleus and cytoplasm of GIV-infected GK cells (Fig. 7). Overall, these results demonstrated that GIV-MCP-mAb-21 could discriminate GIV-infected cells from uninfected ones, suggesting that the mAb can be developed into a diagnostic tool for GIV infection.

332

4. Discussion

322 323 324 325 326 327 328 329 330

333 334 335 336

Monoclonal antibodies are a powerful tool for virus detection and characterization (Tian et al., 2013; Zhang et al., 2013b). In recent years, a large number of mAbs have been developed for diagnosing diseases found in aquaculture (Lai et al., 2002; Shi et al., 2003;

5

Fig. 5. Purification and affinity assay of mouse GIV-MCP-mAb-21. (A) SDS-PAGE of mouse GIV-MCP-mAb-21 purified from ascites resulting from injection of hybridoma 21. M, pre-stained protein marker. (B) Western blot assay for measuring GIV-MCP-mAb-21 titer against GIV-MCP, with 1 ␮g of PGIVP loaded per lane and GIV-MCP-mAb-21 dilutions ranging from 0.05 to 1 ␮g.

Fig. 6. GIV-MCP expression pattern over the course of infection, determined by RTPCR and Western blot. (A) RT-PCR detection of GIV-MCP m-RNA expression with the presence of AraC or CHX in GIV-infected GK cells. (B) Western blot detection of GIV-MCP protein expression in GIV-infected GK cells.

Cote et al., 2009; Hou et al., 2011; Aamelfot et al., 2013; Patil et al., 2013; Siriwattanarat et al., 2013). The present report describes the development and characterization of several mouse mAbs specific to the MCP of GIV. The process involved generating six mouse IgG1 mAbs against GIV-MCP using the recombinant GIV-MCP-His fusion protein. Subsequently, ELISA and Western blot assays were used to confirm the specificity of these mAbs. Among the mAbs, GIV-MCPmAb-21 was most reactive in ELISA, and was further shown to be effective reagent to detect the intracellular localization of GIV-MCP.

Fig. 4. Screening and isotyping of six positive hybridoma cells. (A) Screening of six positive hybridoma cells by Western blot. M, pre-stained protein marker; V, purified grouper iridovirus; R, purified recombinant GIV-MCP-His. (B) Isotype detection of six GIV-MCP monoclonal antibodies by ELISA. Lane C, only secondary antibody.

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

337 338 339 340 341 342 343 344 345

G Model VIRMET 12506 1–7

ARTICLE IN PRESS

6

H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

Fig. 7. Immunofluorescence assay for expression and intracellular localization of GIV-MCP protein using GIV-MCP-mAb-21 in GK cells at 0, 24, and 36 h post-GIV infection.

346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378

For viruses such as GIV, viral gene transcription can be classified as immediate early (IE), early (E), and late (L) according to the gene’s temporal synthesis (Williams, 1996). By definition, the expression of IE genes relies solely on host proteins. IE proteins are often essential for the viral life cycle (Willis and Granoff, 1985; Xia et al., 2010), and frequently have functions related to activating the expression of viral early and late genes (Buisson et al., 1989), altering the host gene functions, and eliminating host immune defense (Holley-Guthrie et al., 1990; Huang et al., 2011). On the other hand, late genes usually encode the viral structural proteins (Wan et al., 2010), and their protein expression often occurs after the viral genome replication. The MCP of iridoviruses is the predominant structural component of the virus and is often a late gene. The MCP gene is commonly used to analyze the phylogenetic relationships among iridoviruses (Do et al., 2005; Lu et al., 2005; Wang et al., 2009; Ohlemeyer et al., 2011). Being the most abundant component of a virion, the iridoviral MCP is often chosen as a vaccine candidate (Kim et al., 2008; Fu et al., 2012; Seo et al., 2013) and a target for detecting iridoviral infection (Huang et al., 2004; Eaton et al., 2007; Chinchar et al., 2009; Wang et al., 2009). In the present study, the temporal expression of GIV-MCP was analyzed by RTPCR in the presence of inhibitors against DNA or protein synthesis. The data support that the GIV-MCP is a late gene during viral replication. With the availability of GIV-MCP-specific mAbs established in this study, we were able to confirm the late expression of GIVMCP in the infected cells. The data also demonstrated the validity of the GIV-MCP-mAb-21 as a useful analytical tool for GIV infection. Previously, several mAbs have been raised against purified SGIV and were used successfully to identify the subcellular localization of two viral proteins, VP100 and VP117, whose molecular mass was estimated to be 100 and 117 kDa, respectively. Both VP100 and VP 117 colocalize with the virus assembly sites in the cytoplasm of SGIV-infected grouper cells, indicating potential involvement

of the two proteins with SGIV assembly (Shi et al., 2003). In the present study, the immunofluorescence assay with GIV-MCP-mAb21 showed that GIV-MCP localized in not only the cytoplasm but also nucleus. The MCP is known as a structural protein of virion, yet its presence in nucleus might indicate additional role of MCP during GIV replication. The significance of the additional unknown role of GIV-MCP during viral replication will be further investigated in our future studies. In addition, since the GIV-MCP shares 99% homology with its counterpart in SGIV, it is likely that mAb-21 could cross-react with the major capsid protein of SGIV and perhaps other ranaviruses. It will be interesting to assay the species specificity of the six newly established mAbs in future study. The data will assist in establishing diagnostic reagent that is specific to GIV only or closely related ranaviruses. In conclusion, the present report describes the generation of six mouse mAbs specific to GIV-MCP. These mAbs were shown to be an effective diagnostic tool for GIV infection. In particular, we have demonstrated that GIV-MCP-mAb-21 is a valid tool for studying of the function of GIV-MCP during viral infection. These novel GIVMCP mAbs will also benefit future studies of GIV pathogenicity.

Acknowledgement This study was supported by Grant No. NSC 102-2815-C-197Q2 008-B from the National Science Council, Taiwan.

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. J. Anat. 222, 547–557.

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398

399

400 401

402

403 404 405 406

G Model VIRMET 12506 1–7

ARTICLE IN PRESS H.-Y. Lin et al. / Journal of Virological Methods xxx (2014) xxx–xxx

407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 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. J. Virol. 63, 5276–5284. Chi, S.C., 1997. The Investigation of Viral Disease Among Cultured Groupers in Southern Taiwan., pp. 59–69. Chinchar, V.G., Essbauer, S., He, J.G., Hyatt, A., Miyazaki, T., Seligy, V., Williams, T., 2005. Family Iridoviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: XIII Report of the International Committee on Taxonomy of Viruses. Elsevier, London, pp. 163–175. Chinchar, V.G., Hyatt, A., Miyazaki, T., Williams, T., 2009. Family Iridoviridae: poor viral relations no longer. Curr. Top. Microbiol. Immunol. 328, 123–170. 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. J. Fish Dis. 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. J. Fish Dis. 32, 989–996. Do, J.W., Cha, S.J., Kim, J.S., An, E.J., Lee, N.S., Choi, H.J., Lee, C.H., Park, M.S., Kim, J.W., Kim, Y.C., Park, J.W., 2005. Phylogenetic analysis of the major capsid protein gene of iridovirus isolates from cultured flounders Paralichthys olivaceus in Korea. Dis. Aquat. Organ. 64, 193–200. 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. Virol. J. 4, 11. Fofana, I., Xiao, F., Thumann, C., Turek, M., Zona, L., Tawar, R.G., Grunert, F., Thompson, J., Zeisel, M.B., Baumert, T.F., 2013. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell–cell transmission. PLOS ONE 8, e64221. Fu, X., Li, N., Lai, Y., Liu, L., Lin, Q., Shi, C., Huang, Z., Wu, S., 2012. Protective immunity against iridovirus disease in mandarin fish, induced by recombinant major capsid protein of infectious spleen and kidney necrosis virus. Fish Shellfish Immunol. 33, 880–885. 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. J. Fish Dis. 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. J. Virol. 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. Virol. Sin. 26, 260–266. Huang, C., Zhang, X., Gin, K.Y., Qin, Q.W., 2004. In situ hybridization of a marine fish virus, Singapore grouper iridovirus with a nucleic acid probe of major capsid protein. J. Virol. Methods 117, 123–128. 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. J. Gen. Virol. 92, 1292–1301. Kim, T.J., Jang, E.J., Lee, J.I., 2008. Vaccination of rock bream, Oplegnathus fasciatus (Temminck & Schlegel), using a recombinant major capsid protein of fish iridovirus. J. Fish Dis. 31, 547–551. Lai, Y.S., Chiu, H.C., Murali, S., Guo, I.C., Chen, S.C., Fang, K., Chang, C.Y., 2001a. 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). J. Fish Dis. 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. J. Fish Dis. 26, 31–42. Lai, Y.S., Murali, S., Chiu, H.C., Ju, H.Y., Lin, Y.S., Chen, S.C., Guo, I.C., Fang, K., Chang, C.Y., 2001b. Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue. J. Fish Dis. 24, 299–309. 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

7

of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus. J. Fish Dis. 23, 379–388. Langdon, J.S., Humphrey, J.D., 1987. Epizootic haematopoietic necrosis, a new viral disease in redfin perch, Perca fluviatilis L., in Australia. J. Fish Dis. 10, 289–297. Lu, L., Zhou, S.Y., Chen, C., Weng, S.P., Chan, S.M., He, J.G., 2005. Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides. Virology 339, 81–100. 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). J. Fish Dis. 25, 91–100. Ohlemeyer, S., Holopainen, R., Tapiovaara, H., Bergmann, S.M., Schutze, H., 2011. Major capsid protein gene sequence analysis of the Santee-Cooper ranaviruses DFV, GV6, and LMBV. Dis. Aquat. Organ. 96, 195–207. 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 tiger shrimp Penaeus monodon. J. Fish Dis. 36, 753–762. 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. Dis. Aquat. Organ. 53, 1–9. Schuh, J.C.L., Shirley, I.G., 1990. Viral hematopoietic necrosis in an angelfish (Pterophyllum scalare). J. Zoo Wildlife Med. 21, 95–98. Seo, J.Y., Chung, H.J., Kim, T.J., 2013. Codon-optimized expression of fish iridovirus capsid protein in yeast and its application as an oral vaccine candidate. J. Fish Dis. 36, 763–768. Shi, C., Wei, Q., Gin, K.Y., Lam, T.J., 2003. Production and characterization of monoclonal antibodies to a grouper iridovirus. J. Virol. 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. Arch. Virol. 158, 967–979. Sung, C.H., Chi, S.C., Huang, K.C., Lu, J.K., 2010. Rapid detection of grouper iridovirus by loop-mediated isothermal amplification. J. Mar. Sci. Technol. 18, 568–573. Tian, Y., Chen, W., Yang, Y., Xu, X., Zhang, J., Wang, J., Xiao, L., Chen, Z., 2013. Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody. Appl. Microbiol. Biotechnol. 97, 1553–1560. Tidona, C.A., Schnitzler, P., Kehm, R., Darai, G., 1998. Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution? Virus Genes 16, 59–66. Toi, M., Takada, M., Bando, H., Toyama, K., Yamashiro, H., Horiguchi, S., Saji, S., 2004. Current status of antibody therapy for breast cancer. Breast Cancer 11, 10–14. 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. J. Virol. 79, 2010–2023. 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. Arch. Virol. 155, 351–359. Wang, C.S., Chao, S.Y., Ku, C.C., Wen, C.M., Shih, H.H., 2009. PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan. J. Fish Dis. 32, 543–550. Wang, W., Yang, P., Zhong, Y., Zhao, Z., Xing, L., Zhao, Y., Zou, Z., Zhang, Y., Li, C., Li, T., Wang, C., Wang, Z., Yu, X., Cao, B., Gao, X., Penninger, J.M., Wang, X., Jiang, C., 2013. Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury. Cell Res. 23, 577–580. Williams, T., 1996. The iridoviruses. Adv. Virus Res. 46, 345–412. Williams, T., Barbosa-Solomieu, V., Chinchar, V.G., 2005. A decade of advances in iridovirus research. Adv. Virus Res. 65, 173–248. Willis, D.B., Granoff, A., 1985. trans activation of an immediate-early frog virus 3 promoter by a virion protein. J. Virol. 56, 495–501. 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 Res. 147, 30–39. Zhang, Y., Wu, S., Wang, J., Wernike, K., Lv, J., Feng, C., Zhang, J., Wang, C., Deng, J., Yuan, X., Lin, X., 2013a. Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein. Protein Expr. Purif. 92, 1–8. Zhang, Z., Li, X., Yi, W., Li, S., Hu, C., Chen, A., 2013b. A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection. Clin. Biochem. 46, 1105–1110.

Please cite this article in press as: Lin, H.-Y., et al., Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein. J. Virol. Methods (2014), http://dx.doi.org/10.1016/j.jviromet.2014.04.013

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

Development and application of a monoclonal antibody against grouper iridovirus (GIV) major capsid protein.

The major capsid protein (MCP) is a main structural protein of iridoviruses, and is used as a marker for the identification, differentiation and class...
2MB Sizes 0 Downloads 4 Views