EcVig, a novel grouper immune-gene associated with antiviral activity against NNV infection

Developmental and Comparative Immunology 43 (2014) 68–75

Contents lists available at ScienceDirect

Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci

EcVig, a novel grouper immune-gene associated with antiviral activity against NNV infection Ying-Chun Yeh a, Yi-Jiou Hsu a, Yi-Min Chen a, Han-You Lin a, Huey-Lang Yang a,b, Tzong-Yueh Chen a, Han-Ching Wang a,⇑ a b

Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, ROC Merit Ocean Biotech Inc., Tainan, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 7 October 2013 Revised 29 October 2013 Accepted 29 October 2013 Available online 6 November 2013 Keywords: VHSV induced gene (Vig) Orange-spotted grouper (Epinephelus coioides) Nervous necrosis virus (NNV)

a b s t r a c t VHSV-induced genes (VIGs) were first identified in rainbow trout (Oncorhynchus mykiss) and subsequently isolated in a variety of fish. Recent studies have shown that most VIGs have immunological functions against pathogenic infections. However, most research has focused on Vig1, such that our present understanding of these genes in other fish species remains limited. This study isolated a homologue of the uncharacterized O. mykiss Vig-B319 (EcVig) from orange-spotted grouper (Epinephelus coioides). Genomic organization suggests that four EcVig isoforms (EcVig A–D), are generated through alternative splicing. Due to the encoding of 2 immunoglobulin (Ig) domains, the EcVig protein can be considered a member of the immunoglobulin superfamily. The expression of EcVig increased 3 days after hatching (dph) and peaked at 9 dph. This pattern is similar to that displayed by EcMx, an important grouper antiviral gene. Additionally, a tissue tropism assay revealed that EcVig A is the major EcVig isoform present in the tissues considered by this study, with the expression of EcVig A exceeding that of EcVig B. We subsequently investigated whether EcVig expression was induced by the viral pathogen nervous necrosis virus (NNV) or the bacterial pathogen Vibrio anguillarum. Following injection with NNV, the expression levels of EcVig showed significant up-regulation. Conversely, a significant reduction was observed in EcVig expression in brain samples collected from V. anguillarum injected grouper. The overexpression of EcVig A suppressed the replication of NNV in grouper GF-1 cell lines, suggesting that EcVig is an important antiviral factor in the grouper immune responses. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Non-specific innate immunity is the primary defense against pathogens, such as viruses, bacteria, and parasites. Even among lower vertebrates, such as fish, the first line of antiviral defense depends on Type I (a/b) interferons (IFNs) (Randall and Goodbourn, 2008). Following secretion by virus infected cells, IFNs bind to the IFN(a/b) receptor (IFNAR) on the surface of healthy cells, which activates hundreds of interferon stimulated genes (ISGs). This in turn triggers anti-viral defenses and reduces unfavorable pathological outcomes (Bonjardim et al., 2009; Koyama et al., 2008; Chen et al., 2013). To understand the interactions between pathogenic viruses and fish, O’Farrell et al. (2002) used suppression subtractive hybridization (SSH) techniques and found a group of genes which were up-regulated following infection with viral hemorrhagic ⇑ Corresponding author. Address: Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan, ROC. Tel.: +886 6 2757575x65603 810; fax: +886 6 276 6505. E-mail address: [email protected] (H.-C. Wang). 0145-305X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2013.10.014

septicemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss). However, lacking a sequence homology, they were described as VHSV-induced genes (VIGs) (O’Farrell et al., 2002). Those researchers further determined that most VIGs could be induced by rainbow trout interferons, which suggests that these genes are also ISGs. Later research indicated that a number of these VIGs play important roles in anti-viral response and anti-bacterial defense (Lee et al., 2013). For instance, rainbow trout Vig1 is a homologue of human viperin (virus inhibitory protein, endoplasmic reticulumassociated, interferon-inducible); thus, it can be triggered by viruses, LPS, poly (I:C) or IFNs (Chin and Cresswell, 2001; Sun and Nie, 2004; Mattijssen and Pruijn, 2012). Due to their antiviral activity, it is not surprising that homologues of VIGs have been isolated from other fish species, including siniperca, coreoperca, sockeye salmon (Oncorhynchus nerka), Atlantic salmon (Salmo salar), and zebrafish (Danio rerio) (Chen et al., 2010; Purcell et al., 2009; Lukacs et al., 2010; Levraud et al., 2007). However, apart from Vig1, antiviral and antimicrobial activities triggered by VIGs have not been elucidated. Orange-spotted grouper (Epinephelus coioides) is an important aquaculture fish species in subtropical/tropical countries.

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

69

Therefore, this present study isolated and characterized the fulllength cDNA sequence of a novel Vig gene from E. coioides, named EcVig. Notably, this gene is homologous with Vig-B319 in rainbow trout. We then investigated the expression of EcVig when groupers were administered NNV (nervous necrosis virus) and Vibrio anguillarum. Finally, we used a transient transfection system to determine whether EcVig presents antiviral activity against NNV infection.

Two EcVig specific primer sets (pcDNA3-VigA_F/EcVig_50 -1_R and EcVig_T_qPCR_F/EcVig_skip _R) were used to amplify the N- and C- terminal EcVig coding regions, respectively (Fig. 1A; Table 1). The PCR products were then cloned into RBC T&A cloning vectors and sequenced.

2. Materials and methods

The Simple Molecular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/) was used to identify the domain architecture of EcVig isoforms, while the Eukaryotic Linear Motif resource for Functional Sites in Proteins (http://elm.eu.org/) was used to predict signal peptide regions as well as indicate domains and motifs.

2.1. Experimental animals and cells The species of interest for this study was orange-spotted grouper (E. coioides). Thus, disinfected fertilized eggs and specific-pathogen-free (SPF) fry between 1–60 days post hatch (dph) were purchased form Merit Ocean Biotech Inc., Tainan, Taiwan. Cultivation conditions have previously been described (Lam et al., 2011; Yeh et al., 2012). 2.2. Cloning of full-length EcVig cDNAs Juvenile fish specimens of various ages (5, 14 and 28 dph) were permeabilized in REzolTMC&T reagent (Protech Technology, Taiwan). Total RNA was then extracted for cDNA synthesis. Specifically, 50 cDNA and 30 cDNAs for RACE (rapid amplification of cDNA ends) were synthesized using the SMART™ RACE cDNA Amplification Kit (Clontech), while general cDNAs for 30 RACE, gene cloning, RT-PCR, and real-time PCR were synthesized using Superscriptase II (Invitrogen) and Anchor dTv primers (Table 1). Specific primers were derived from previously published partial cDNA sequences of VHSV-induced protein in E. coioides (GenBank accession No. FJ438491.1). We then employed these primers to isolate Vig homologues from E. coioides. RACE PCRs of the N-terminal and C-terminal sequences of EcVig were subsequently performed using the primer sets NUP/EcVig_T_qPCR_R and EcVig_T_qPCR_F/ Anchor dTv, respectively (Fig. 1A; Table 1). The PCR products were then cloned into RBC T&A cloning vectors (RBC Bioscience, Taiwan), sequenced, and assembled using Genedoc software. 2.3. Cloning a partial genomic sequence of the EcVig gene Total genomic DNA was extracted from grouper (28 dph) using the DTAB/CTAB DNA extraction kit (GeneReach Biotech. Corp.).

2.4. Domain search of EcVigs

2.5. Expression of EcVigs in larval development stages of E. coioides Fertile eggs and fry were collected at regular time intervals between 1–27 dph using previously described methods (Yeh et al., 2012). Total RNA was extracted using REzolTMC&T reagent, and cDNA was synthesized using Superscriptase II (Invitrogen) and Anchor dTv primer (Table 1). To investigate EcVig expression, we designed specific primers for real-time quantitative PCR (real-time PCR) according to the housekeeping gene EcEF1a (EcEF1a_qPCR_F/EcEF1a_qPCR_R), total EcVig (EcVig_T_qPCR_F/EcVig_T_qPCR_R), a biomarker of fish antiviral immune-related gene myxovirus-resistance gene (EcMx, EcMx_qPCR_F/ EcMx_qPCR_R). All primers are listed in Table 1. Real-time PCR was performed using a Bio-Rad CFX Connect™ real-time PCR detection system with KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, USA). Expressions of total EcVig and EcMx relative to EcEF1a were measured using the 2ðD DCtÞ method. Relative expression values are presented as 2ðD DCtÞ normalized against the basal expression of the egg. Mean data values are presented ± SD (standard deviation) for each quadruplicate sample. 2.6. Tissue tropism of EcVig mRNAs Due to the small size of fry younger than 30 dph, we collected tissue samples from groupers aged 60 dph (size: 5–6 cm). Fifteen types of tissue were collected: muscle, blood, gill, thymus, heart, head kidney, trunk kidney, brain, eye, optic nerve, spleen, liver, stomach, pancreas, and intestine. For each tissue type, four pooled

Table 1 PCR primers used in this study. Primer name

Primer sequence (50 –30 )

Usage

EcVig_T_qPCR_F EcVig_T_qPCR_R EcVig_50 -1_R EcVig_skip_R EcEF1a_qPCR_F EcEF1a_qPCR_R EcMx_qPCR_F EcMx_qPCR_R NNV_RNA2_qPCR_F NNV_RNA2_qPCR_R EcEF1a_F EcEF1a_R pcDNA3-VigA_F

50 -CCAGGTGAGGACTTAGTTTTGGA-30 50 -TGCCCAGGAAGACCACTCTT-30 50 - CATCACCGAGGCGTTTCTTC-30 50 -GAACAGACAAGATGAGAAGA-30 50 -GATGGGCAAGGGCTCCTT-30 50 -CGCTCGGCCTTCAGTTTGT-30 50 -GCCAAGATTGAAGCCATTAAGC-30 50 -AACTGGGTCCTCAGCATGGA-30 50 -GACGCGCTTCAAGCAACTC-30 50 -CGAACACTCCAGCGACACAGTA-30 50 -GTCAACAAGATGGACTCCAC-30 50 -AGGGTGGTTCAGGATGATGA-30

Real-time PCR and cloning of total EcVig

50 -GGGGATCCATGAAAAACGTCCGAAGGCGAC-30

Cloning of pcDNA3-VigA

pcDNA3-VigA_R

50 -CCGAATTCGAGGAGATGAAGAATTTGGAAAC-30

pcDNA3-EGFP_F

50 -GGGGATCCATGGTGAGCAAGGGCGAGGAG-30

pcDNA3-EGFP_R

50 -CCGAATTCCTTGTACAGCTCGTCCATGCC-30 50 -CCCATATGTCTTCCCTGTCCTCCTCCAC-30 50 -CCGTCGACCTCCACTGAGAGCTGCACGG-30 50 -AAGCAGTGGTATCAACGCAGAGT-30 50 -GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-30 50 -NNNNNN-30

pET28b-Vig_NS_F pET28b-Vig_NS_R NUP Anchor-dTv Random hexamer

Genomic sequence of EcVig Genomic sequence and RT-PCR for EcVig A and B Real-time PCR for EcEF1a Real-time PCR of EcMx Real-time PCR of NNV RNA2 RT-PCR of EcEF1a

Cloning of pcDNA3-EGFP Cloning of pET28b-Vig RACE PCR cDNA synthesis cDNA synthesis

70

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75 E2

(A)

5’UTR SP

Cloning of the full-length cDNA Cloning of the partial genomic structure

Ig 1N

Ig 1C E1

Ig 2

Ig 2

E3 E4 E5

3’UTR

EcVig_T_qPCR_R

NUP

Anchor dTv

EcVig_T_qPCR_F

pcDNA3-VigA_F

EcVig_5’-1_R EcVig_T_qPCR_F

EcVig_skip _R

Primers for RT-PCR

EcVig_T_qPCR_F

EcVig_skip _R

Primers for Real-time PCR

EcVig_T_qPCR_F

Cloning in plasmid vectors

EcVig_T_qPCR_R

pET28b-Vig_NS_R

pET28b-Vig_NS_F

pcDNA3-VigA_R

pcDNA3-VigA_F

(B)

EcVig A

SP

Ig 1N

Ig 1C E1

Ig 2

Ig 2

E3

E5

*

SP

Ig 1

MKNVRRRLWFFIFLQCVLVGSLVDSSSLSSSTQSLSITSKVGNQAVLPCSWKKRLGDVALPSCHIQWATPADTVFELQGEQKWQAEEFEG

90

E1 RVEVPKEKLGSGDCSLIINDVQIGDTGRYESFMVVEGVRSIKTRVFIQSVKFTVFDHKVLQSKGPGEDLVLDLYTRHSLRVVFLGRNSSV

SKI1

EcVig B

180

FHA

Ig 2

E3

E5

WSDLWMRGDANSERLQKHPLNEQLTIMNLKSSDEGTYKVLDEHGLAVSTVQLSVEENSTALKHHQIHLENPVPTGDAVKSSSSVLLILSV

270

LVMSFQILHLL-

282

SP

Ig 1N

Ig 1C E1

Ig 2

Ig 2

E3 E4 E5

*

SP

Ig 1

MKNVRRRLWFFIFLQCVLVGSLVDSSSLSSSTQSLSITSKVGNQAVLPCSWKKRLGDVALPSCHIQWATPADTVFELQGEQKWQAEEFEG

E1

RVEVPKEKLGSGDCSLIINDVQIGDTGRYESFMVVEGVRSIKTRVFIQSVKFTVFDHKVLQSKGPGEDLVLDLYTRHSLRVVFLGRNSSV SKI1 FHA

Ig 2

E3

E4

WSDLWMRGDANSERLQKHPLNEQLTIMNLKSSDEGTYKVLDEHGLAVSTVQLSVEENSTALKHHQIHLENPVPTGEKIMFLFWQRVKKKK SKI1 NLS

E5

KILICNNPLSPPSDVSQVMLSKAAVQFFSSCLFLS-

90

180

270

305

E2

EcVig C

SP

Ig 1N

Ig 1C E1

Ig 2

Ig 2

E3

E5

*

SP

Ig 1

MKNVRRRLWFFIFLQCVLVGSLVDSSSLSSSTQSLSITSKVGNQAVLPCSWKKRLGDVALPSCHIQWATPADTVFELQGEQKWQAEEFEG

E1

RVEVPKEKLGSGDCSLIINDVQIGDTGRYESFMVVEGVRSIKTRVFIQSVKFTVFDHKVLQSKGPGEDLVLDLYTRHSLRVVFLGRNSSV SKI1 FHA

Ig 2

EcVig D

E2

E3

E5

90

180

WSDLWMRGDANSERLQKHPLNEQLTIMNLKSSDEGTYKVLDEHGLAVSTVQLSVEVFTVSQKTPQLSNTTRSIWKIQYQQVMLSKAAVQF

270

FSSCLFLS-

278

SP

Ig 1N

Ig 2

Ig 2

E3

E5

SP

* Ig 1

MKNVRRRLWFFIFLQCVLVGSLVDSSSLSSSTQSLSITSKVGNQAVLPCSWKKRLGDVALPSCHIQWATPADTVFELQGEQKWQAEEFEG

Ig 2

RVEVPKEKLGSGDCSLIKFTVFDHKVLQSKGPGEDLVLDLYTRHSLRVVFLGRNSSVWSDLWMRGDANSERLQKHPLNEQLTIMNLKSSD FHA

E3

90

180

E5

EGTYKVLDEHGLAVSTVQLSVEENSTALKHHQIHLENPVPTGDAVKSSSSVLLILSVLVM-

240

Fig. 1. Schematic diagram outlining the organizational structure of the EcVig transcript. (A) The locations of primers used for cloning, RT-PCR, and real-time PCR relative to the gene structure of EcVig. Gray boxed exons may be included in the mRNA or skipped. A solid line indicates a confirmed intron region, and a dotted line represents a predicted intron region that has not been confirmed by PCR. E signifies an element. (B) EcVig splicing possibilities resulted in four alternative isoforms, EcVig A–D. EcVig can be coded when either E2 or E4 is skipped, resulting in the translation of E3 or E5 in a different reading frame. Abbreviations are as follows: SP, signal peptide; Ig, immunoglobulin domain; FHA, FHA binding motif; SKI1, Subtilisin/kexin isozyme-1 (SKI1) cleavage site; and NLS, nuclear localization signal.

samples (3 groupers for each pool) were collected and subjected to total RNA extraction using REzolTMC&T reagent as well as cDNA synthesis using Superscriptase II (Invitrogen) and Anchor dTv primers (Table 1). Real-time PCR was performed with the primer sets EcEF1a_qPCR_F/EcEF1a_qPCR-R, EcVig_T_qPCR_F/EcVig_T_qPCR_R, and EcMx_qPCR_F/EcMx_qPCR_R using a Bio-Rad CFX Connect™ real-time PCR detection system with KAPA SYBR FAST qPCR Master Mix. Relative expression values are presented in accordance with the 2ðD DCtÞ method. To distinguish distribution

of EcVig A and EcVig B in the various tissues, RT-PCR was performed using EcVig A- or EcVig B-specific primer sets (EcVig_T_qPCR_F/EcVig_skip _R) (Fig. 1A). EcEF1a (EcEF1a_F/EcEF1a_R) was used as an internal host control. 2.7. EcVig antibody production A PCR fragment representing partial EcVig containing the Ig1– Ig2 region was amplified using the primer set (pET28b-Vig_NS_F/

71

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

To determine EcVig protein distribution in various fish tissues, nine types tissue (gill, thymus, heart, head kidney, trunk kidney, brain, eye, spleen, stomach) were collected from groupers (60 dph, size: 5–6 cm) and lysed in cold 0.33 phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) with protein inhibitors (Roche). The concentration of each protein lysate was determined using the Bradford method with Protein Assay reagent (Bio-Rad). The procedure for Western blotting was as follows: Each extract (20 lg) was separated in 12% SDS–PAGE gel. Following electrophoresis, SDS–PAGE was transferred onto a PDVF membrane for Western blotting. Following incubation with blocking solution (1% skim milk in TBST solution [50 mM Tris, 500 mM NaCl, 0.1% Tween 20 at pH 7.5]), the membrane was blotted using anti-EcVig polyclonal antibodies in TBST solution. Incubation and blotting procedures were performed overnight at room temperature. We washed the membranes with TBST solution 3 times (5 min per wash), and subsequently incubated them with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) in TBST solution for 1 h at room temperature. Membranes were then subjected to several additional washing steps. After developing signals using ECL Plus™ detection agents (PerkinElmer Inc.), the protein bands were visualized using a Western Lightning Plus ECL detection system (PerkinElmer Inc.). The expression of actin protein was used as an internal host control. Signal intensities of EcVig and Actin were quantified using Image J computer software. 2.9. NNV inoculation and the challenge experiment NNV inoculation was performed as previously described (Yeh et al., 2012). Briefly, after being isolated from a batch of NNV-infected E. coioides, NNV was propagated in a grouper fin cell (GF1) culture system. A cytopathic effects (CPE) assay was used to determine the virus titer in TCID50 (tissue culture infections dose 50). Through intraperitoneal injection, groupers (37 dph, size: 2 cm) were administered 1  107 TCID50 of NNV, while the control groupers were injected with L15 medium. We collected six pooled samples of brain tissue at various time points following injection. Each pooled sample was obtained from three groupers. Total RNA was extracted as described above and then subjected to cDNA synthesis using Superscriptase II and random hexamer primer (Table 1). Real-time PCR analysis was performed as described above. 2.10. V. anguillarum inoculation and the challenge experiment The stock inoculation of V. anguillarum serotype O1, the causative agent of haemorrhagic septicaemia in marine fish, was prepared from a batch of diseased E. coioides. Groupers (50 dph, size: 5–6 cm) were administered 1  106 colony forming units (CFU) of V. anguillarum serotype O1, while the control groupers were injected with pyrogen free water. From each group, six pooled samples of brain tissue were collected, with each pooled sample obtained from 3 groupers. The procedures for RNA extraction, cDNA synthesis, and real-time PCR were as described above.

To determine the anti-viral response of rEcVig A, a transient overexpression was performed using grouper fin GF-1 cells (Chi et al., 1999). Briefly, to construct the transient expression vector pcDNA3/EcVigA, full-length EcVig A was amplified using the primer set (pcDNA3-VigA_F/pcDNA3-VigA_R; Fig. 1A) listed in Table 1. Following digestion with BamHI and EcoRI, the treated DNA fragment was cloned into pcDNA3.1/V5-His A plasmid (pcDNA3). GF1 cells in 24-well plates were then transfected with 0.2 lg pcDNA3/EcVigA (or with pcDNA3/EGFP control plasmid) using 1.6 ll enhancer and 5 ll Effectene transfection reagent (QIAGEN) per well. At 3 days post transfection, the cells were infected with NNV. At 2 h post NNV infection, cells were washed and incubated using L15 medium. Virus-infected transfected cells were collected at various time points and subjected to total RNA extraction and cDNA synthesis, as described above. Expressions of the EcVig gene and the NNV genome were investigated using real-time PCR.

3. Results 3.1. Characterization of EcVig After cloning and sequencing, four EcVig isoforms, EcVig A, EcVig B, EcVig C, and EcVig D, were isolated (Fig. 1B). Analysis of partial genomic sequences of the EcVig gene suggested that these four isoforms are generated from a single genome locus through alternative splicing. As shown in Fig. 1, three elements can be skipped, including E1 containing the C-terminal of the Ig1 domain, E2, and E4. We note that EcVig D lost all alternative elements. Expression data revealed that EcVig A and EcVig B are the predominant alternatively spliced isoforms of the EcVig gene (data not shown). The amino acid sequence of EcVig showed the highly sequence identity to rainbow trout Vig-B319 (GenBank accession No: NP_001233274). Using the ELM functional site prediction resource, a putative signal peptide was found at aa 1–25. Except for EcVig D, all EcVig Fold of EcVig expression

2.8. Western blot analysis of EcVig protein in various grouper organs

2.11. Anti-viral response of rEcVig A in pcDNA3/EcVigA transfected GF1 cells

Fold of EcMx expression

pET28b-Vig_NS_R; Fig. 1A) listed in Table 1. Following digestion with restriction enzymes NdeI and SalI, the treated DNA fragment was cloned into pET-28b (+). Recombinant plasmid pET-28b (+)EcVig A was transformed into BL21 (DE3). For the recombinant EcVig A protein expression and the generation of polyclonal hyperimmune serum against rEcVigs in New Zealand white rabbits were followed the procedure described previously (Hung et al., 2013).

200

(A) EcVig

*

150

*

* *

100

*

*

*

*

50 0

400

E

1

2

(B)

*

*

*

*

3

4

5

6

* 7

8

9

10 11 12 13 20 27 day post hatch (dph) *p<0.05

*

*

EcMx

* 300

*

200

*

* *

100 0

E

1

*

*

*

*

*

2

3

4

5

6

* *

7

8

9

10 11 12 13 20 27 day post hatch (dph) *p<0.05

Fig. 2. EcVig mRNA expression detected by real-Time PCR during grouper larval development (egg [E] and 1–27 days post hatch [dph]). (A) Total EcVig expression during grouper larval development. (B) Expression of Mx, an antiviral factor, during grouper larval development. Data represents the fold change of each gene following normalization relative to EF1a and the basal expression level at egg. ⁄p < 0.05 relative to gene expression in eggs.

72

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

mRNA expression (gene / EF1α)

(A) 0.25

EcVig EcMx

0.2

*

0.15

* *

0.1 0.05

*

*

*

* *

0

I

St P

L

S

O

E

H HK TK B

T

M BL G

* Organ *p<0.05

(B)

EcVig A (Ig2+E3+E5 ) Ig 2

E3

EcVig_T_qPCR_F

1

2

*

E5

Ig 2

E3 E4 E5

EcVig_skip _R EcVig_T_qPCR_F

M

Organ Sample EcVig

EcVig B (Ig2+E3+E4+E5 )

BL 3

4

1

2

3

EcVig_skip _R

G 4 1

2

H

T

3

4

1

*

2

3

4

1

2

3

4 EcVig B EcVig A

EcEF1α

HK

Organ Sample

1

2

TK 3

1

2

B

3

4

1

2

E 3

4

1

2

O 3

4

1

2

3 EcVig B

EcVig

EcVig A

EcEF1α

S

Organ Sample EcVig

1

2

ST

L 3

4

1

2

3

4

1

2

3

P 4

1

2

I 3

4

1

2

3 EcVig B EcVig A

EcEF1α

(C)

(D) Organ G rP E ST

34 26

H HK TK

B

E

S

ST

B

E

S

ST Organ

Vig Actin Relative expression of EcVIG / Actin

72 55 43

T

2.5 2 1.5 1 0.5 0

EcVIG

G

T

H

HK TK

Fig. 3. EcVig expression levels in various grouper organs. (A) Real-time PCR results for EcVig and EcMx mRNA expression levels in various grouper organs. Samples from muscle [M], blood [BL], gill [G], thymus [T], heart [H], head kidney [HK], trunk kidney [TK], brain [B], eye [E], optic nerve [O], spleen [S], liver [L], stomach [St], pancreas [P], and intestine [I] were amplified using EcVig- and EcMx-specific primer sets. Relative gene expression was calculated using the 2D DCt method, in which the threshold cycle of EcVig normalized with respect to EF1a. Asterisks indicate a significant difference between the expression levels of EcVig and EcMx. (B) RT-PCR results for EcVig A and EcVig B in various grouper organs. Compared with EcVig B, EcVig A was the major expressed isoform. (C) EcVig protein was detected using anti-EcVig polyclonal antibodies. rP: recombinant EcVig A from E. coli expression system as a positive control; E: protein lysate from eye; ST: protein lysate from stomach. (D) Western blotting results for EcVig protein expression levels in various grouper organs. The abbreviation of each tissue type is as described above. The expression of Actin protein was used as an internal host control. The EcVig/Actin ratio for each tissue type was quantified using Image J software and presented in plotted data.

coding regions contained two entire immunoglobulin domains (Ig1–2). Interestingly, cleavage sites of proprotein convertase subtilisin (SKI1) were found in two of these skipping elements, E1 and E4, while a putative nuclear localization signal (NLS) was also observed in E4. A single Forkhead-associated (FHA) domain, a phosphopeptide recognition domain, was found in E1.

3.2. mRNA expression of EcVigs during larval development stages To investigate the expression of EcVigs during grouper larval development, we designed specific primer sets for total EcVig using real-time PCR analysis (Table 1). As shown in Fig. 2A, expression of total EcVig mRNA in eggs and larvae (1–27 dph) began at 3 dph,

73

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

EcVig

Fold of gene expression

(A)

EcMx

*

50 40

40

*

30

30 20

* 0

1

* 2

10 3

7

14 dpi

*

*

20

*

10 0

NNV RNA2

50

*

0

0

1

* 2

3

106

6

106

4

106

2

106 0

14 dpi

7

*

0

*

*

*

1

2

3

* 7

14 dpi

dpi: day post injection

NNV

L15

*

8

*p<0.05 relative to L15

Fold of gene expression

(B) EcMx

EcVig 2 1.5 1 0.5 0

*

0

1

*

*

3

5

dpi

Pyrogen free water V. anguillarum

5 4 3 2 1 0

* * 0

1

3

5

dpi

dpi: day post injection *p<0.05 relative to pyrogen free water

Fig. 4. EcVig induced in grouper brain tissue following viral infection. (A) EcVig expression in grouper brain at 0, 1, 2, 3, 7, and 14 days post injection (dpi) with NNV. (B) EcVig expression in grouper brain at 0, 1, 3, and 5 days post injection (dpi) with V. anguillarum. Relative expression of target mRNA was calculated using the 2D DCt method and normalized with respect to EF1a. Asterisks indicate a significant difference between the target mRNA levels and those in the corresponding negative control (p < 0.05).

rapidly increased at 7 dph, reached a peak at 9 dph, and then gradually decreased until 27 dph. A similar expression pattern during larval development was also observed in the grouper immune gene EcMx, a crucial effector of bonyfish innate antiviral responses (Fig. 2B). Based on the similarities in these patterns, we postulate that EcVig is involved in grouper immune responses. 3.3. Tissue tropism of EcVigs To investigate the expression of total EcVigs in various organs/ tissues, we performed real-time PCR, RT-PCR, and Western blotting. As shown in Fig. 3A, significantly higher levels of total EcVig expression were detected in organs/tissues associated with immunity (gill [G], blood [BL], pancreas [P], and thymus [T]), the nervous system (brain [B]), and the digestive system (intestine [I]). Generally, the expression of total EcVig in grouper organs/tissues was higher than that of EcMx. The EcVig gene can generate several isoforms through alternative splicing; therefore, we analyzed the expression of two main isoforms, EcVig A and EcVig B, in 15 tissues using RT-PCR with specific primer sets (Table 1). The RT-PCR amplified product of EcVig A and EcVig B were 350 bp and 447 bp long, respectively. Fig. 3B shows that EcVig A expression was observed in a wider range of tissues than EcVig B expression. We subsequently used specific anti-EcVig polyclone antibodies to detect EcVigs in protein lysate extracted from various grouper organs/tissues. The molecular mass of EcVig was found to be approximately 35 kDa (Fig. 3C). Tissue tropism analysis (Fig. 3D) demonstrated that EcVig protein was present in all tissues. The intensity of the EcVig protein to b-actin found in Western blot analysis was quantified using Image J Software. These results showed that the EcVig protein is mainly expressed in the gill [G], heart [H], brain [B], eye [E], and stomach [ST]. 3.4. EcVig expression following pathogenic infection To investigate whether EcVig is involved in the host immune response, we injected groupers with virus (NNV) and bacteria (V. anguillarum), respectively. As shown in Fig. 4A, after juvenile

groupers (37 dph) were injected with NNV, a significant expression of NNV RNA2 (relative to the medium (L15) control) was detected in the brain at 1 dpi. In swimming groupers, this expression peaked at 7 dpi, and then began to decrease. Expression levels of total EcVigs also increased significantly at 1 dpi, reaching a maximum at 3 dpi. Similarly, expression levels of EcMx also increased significantly at 1 dpi and reached a maximum at 7 dpi. This evidence suggests that EcVig expression can be induced following NNV infection, in turn indicating that EcVig may be important in antiviral response. For bacterial infection, brain tissues of juvenile groupers (50 dph) were collected at various time points following injection with gram-negative V. anguillarum. As shown in Fig. 4B, the expression levels of total EcVigs and EcMx were significantly down-regulated from 1 to 5 dpi, compared with the control group. These results suggest that EcVigs is not necessarily induced by bacterial infection.

3.5. Overexpression of EcVig A reduces the number of NNV copies in GF-1 cells To investigate the anti-viral activity of the major EcVig isoform EcVig A, EcVig A was transiently overexpressed in GF-1 cells, and the cells were then subjected to NNV infection. As shown in Fig. 5A, real-time PCR confirmed the overexpression of rEcVig A in GF-1 cells transfected with plasmid pcDNA3/EcVig A, while the expression of endogenous EcVig was also detected in cells transfected with plasmid pcDNA3/EGFP. Moreover, overexpression of EGFP mRNA was only detected in cells transfected with plasmid pcDNA3/EGFP. To further test the antiviral activity of EcVig A against NNV, we used real-time PCR to determine the growth curves of NNV in control cells, EGFP-overexpressing cells and EcVig A-overexpressing cells (Fig. 5B). The virus growth curves showed that the overexpression of EcVig A in GF-1 cells significantly reduced NNV replication from 12 to 72 h post infection, compared with control groups. These results demonstrate that the overexpression of EcVig A significantly inhibits NNV replication in GF-1 cells.

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

x

74

Fig. 5. Overexpression of EcVig A inhibited virus replication. (A) GF-1 cells were transfected with pcDNA3/EcVig A or pcDNA3/EGFP. Cells that were not transfected were used as a control. Cells were collected at indicated time points and subjected to total RNA extraction and cDNA synthesis. Real-time PCR was then used to determine the expression of EcVig and EGFP. (B) At 3 days post transfection, cells were incubated with NNV for 2 h. After rigorous washing, cells were incubated using L15 medium and collected at indicated time points. It should be noted that i refers to samples collected immediately after NNV incubation and subsequent washing. Each dot represents the mean ± SD from four samples. Using real-time PCR, the number of NNV copies in each group was represented after normalization relative to EF1a and proportional adjustment to the corresponding basal (i) expression level, which was set to 1. Control: cells without transfection. Data was analyzed using one-way ANOVA followed by a Tukey HSD test. Different letters represent a significant difference between each group (P < 0.05). NNV replication was inhibited in the EcVig A overexpression group, compared with the two control groups.

4. Discussion In the VIG family, only vig1 3 in fish is currently well understood (Boudinot et al., 1999, 2001; Liu et al., 2002; Zhang et al., 2007; Langevin et al., 2013). This study characterized the novel antiviral immune gene EcVig in orange-spotted grouper (E. coioides). Sequence analysis suggests that EcVig is closely related to uncharacterized rainbow trout VIG-B139 (GenBank accession No.: NP_001233274), with homologies of 50.2% (EcVig A) and 44.8% (EcVig B). The VIG family is clustered according to virusinducible characteristics, rather than sequence homology; therefore, no universal signature motif, domain, or sequences have been identified between VIGs (O’Farrell et al., 2002). For the domain architecture of EcVig, we identified two immunoglobulin domains, Ig1–2 (Fig. 1). Based on the characteristics of this sequence, we tentatively assigned EcVig to the immunoglobulin superfamily. E. coioides presented at least four EcVig isoforms, EcVigs A–D, in which EcVig A is predominant in each type of grouper tissue. The four EcVigs presented identical 50 UTR and 30 UTR sequences (data not shown). Despite three skipping elements (Ig1C + E1, E2 and E4), there was considerable homology between the amino acid sequences of these four isoforms (Fig. 1B). A partial genomic organization of the EcVig gene demonstrated that two skipping elements,

E2 and E4, also present with other constitutive exons at the same locus (Fig. 1A). This evidence suggests that these four isoforms were generated from the same locus via alternative splicing events: exon skipping of Ig1C + E1 and E4; as well as the alternative 30 splice site (ss) selection of E2 (Keren et al., 2010). Alternative 30 ss selection of E2 and skipping of E4 altered the subsequent reading frame, resulting in the two encoding amino acid sequences of E3 and E5, respectively. Even though the alternative splicing events of E2 and E4 increased the complexity of translated EcVig, no motif/domain was found in either of the two encoding amino acid sequences for E3 and E5. The underlying cellular regulation of alternative splicing events of E2 and E4 requires further study. To investigate the involvement of EcVig in the immune defense of groupers, we used several strategies that compared the expression of EcVig with that of a well-known ISG biomarker, EcMx. First, during grouper larval development from egg to fry at 27 dph, the expression pattern of EcVig mRNA presented a single-peak pattern similar to that of EcMx (Fig. 2). The expression of EcVig and EcMx during larval development showed a different pattern than that of genes involved in physiological development (Yeh et al., 2012). Our experiment on tissue tropism further demonstrated that, compared with EcMx expression levels, EcVig expression was significantly high in nearly all tissues, including immune-related

Y.-C. Yeh et al. / Developmental and Comparative Immunology 43 (2014) 68–75

tissues (blood and thymus), NNV target tissues (brain, eye and optic nerve), and tissues of the digestive system (liver, pancreas and intestine) (Fig. 3A). It should be noted that the expression levels of EcVig were significantly above basal [muscle] levels in all tissues [data not shown]. Additionally, EcVig A (an EcVig spliced isoform) was expressed more highly than that of EcVig B in these tissues (Fig. 3B). Based on these results, we infer that EcVig, particularly EcVig A, may be involved in the immune response of groupers. A time-course study of EcVig expression in grouper infected with the viral pathogen NNV and the bacterial pathogen V. anguillarum further substantiated this hypothesis. Fig. 4A illustrates a significant increase in EcVig and EcMx in brain tissue (a target of NNV) 1 day after NNV inoculation; this up-regulation continued over the following days. These results are consistent with those obtained by Chen et al. (2006), which showed a strong EcMx response during NNV infection. In contrast to tilapia (Oreochromis niloticus) Vig1 can been induced through antiviral and antibacterial activities (Lee et al., 2013), EcVig and Mx was not induced in brain or intestinal tissues after V. anguillarum challenge (Fig. 4B; data not shown). These results suggest that EcVig is involved in grouper responses to viruses. However, in the absence of further assays, it is too early to conclude that EcVig is not involved in grouper immune response against bacterial infection. The IFN system plays an important role in cellular defense against viral infection, ISGs are presumed to possess antiviral activity (Verrier et al., 2011). Among teleosts, Mx has been shown to suppress replication in many viruses (Chen et al., 2008; Fernández-Trujillo et al., 2013). To determine whether EcVig is involved in antiviral immunity, we investigated the replication rate of NNV in over-expressed EcVig A GF-1 cells. EcVig A overexpression significantly decreased the NNV replication rate at 12 hpi and continued to suppress this virus until the end of the study period (72 hpi) (Fig. 5). The observations provide the first evidence of EcVig A involvement in antiviral activity against NNV infection. Our present results suggest that grouper cells may benefit from the presence of EcVig A. Since no direct interaction between EcVig and NNV coat protein was observed by using yeast two-hybrid system (data not shown), further studies will be needed to demonstrate the biological antiviral mechanism triggered by EcVig. Taken together, we conclude that EcVig is a novel grouper antiviral immune factor, which can be induced following viral infection. Efficient EcVig expression helps counter viral replication in these fish, although the underlying mechanisms remain unknown. EcVig is believed to act as an ISG that can be stimulated by IFNs; therefore, the next logical step will be to investigate the relationship between IFNs and EcVigs, as well as to elucidate the upstream- and downstream-pathways of this gene. Acknowledgments This investigation was financially supported by National Science Council grants (NSC 101-2628-B-006 -001 -MY4). References Bonjardim, C.A., Ferreira, P.C., Kroon, E.G., 2009. Interferons: signaling, antiviral and viral evasion. Immunol. Lett. 122, 1–11. Boudinot, P., Massin, P., Blanco, M., Riffault, S., Benmansour, A., 1999. Vig-1, a new fish gene induced by the rhabdovirus glycoprotein, has a virus-induced homologue in humans and shares conserved motifs with the MoaA family. J. Virol. 73, 1846–1852.

75

Boudinot, P., Salhi, S., Blanco, M., Benmansour, A., 2001. Viral haemorrhagic septicaemia virus induces vig-2, a new interferon-responsive gene in rainbow trout. Fish Shellfish Immunol. 11, 383–397. Chen, Y.M., Su, Y.L., Lin, J.H., Yang, H.L., Chen, T.Y., 2006. Cloning of an orangespotted grouper (Epinephelus coioides) Mx cDNA and characterisation of its expression in response to nodavirus. Fish Shellfish Immunol. 20, 58–71. Chen, Y.M., Su, Y.L., Shie, P.S., Huang, S.L., Yang, H.L., Chen, T.Y., 2008. Grouper Mx confers resistance to nodavirus and interacts with coat protein. Dev. Comp. Immunol. 32, 825–836. Chen, D., Guo, X., Nie, P., 2010. Phylogenetic studies of sinipercid fish (Perciformes: Sinipercidae) based on multiple genes, with first application of an immunerelated gene, the virus-induced protein (viperin) gene. Mol. Phylogenet. Evol. 55, 1167–1176. Chen, Y.M., Wang, T.Y., Chen, T.Y., 2013. Immunity to betanodavirus infections of marine fish. Dev. Comp. Immunol. in press. Chi, S.C., Hu, W.W., Lo, B.J., 1999. Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV). J. Fish Dis. 22, 173–182. Chin, K.C., Cresswell, P., 2001. Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus. Proc. Natl. Acad. Sci. USA 98, 15125–15130. Fernández-Trujillo, M.A., García-Rosado, E., Alonso, M.C., Castro, D., Alvarez, M.C., Béjar, J., 2013. Mx1, Mx2 and Mx3 proteins from the gilthead seabream (Sparus aurata) show in vitro antiviral activity against RNA and DNA viruses. Mol. Immunol. 56, 630–636. Hung, H.Y., Ng, T.H., Lin, J.H., Chiang, Y.A., Chuang, Y.C., Wang, H.C., 2013. Properties of Litopenaeus vannamei Dscam (LvDscam) isoforms related to specific pathogen recognition. Fish Shellfish Immunol. 35, 1272–1281. Keren, H., Lev-Maor, G., Ast, G., 2010. Alternative splicing and evolution: diversification, exon definition and function. Nat. Rev. Genet. 11, 345–355. Koyama, S., Ishii, K.J., Coban, C., Akira, S., 2008. Innate immune response to viral infection. Cytokine 43, 336–341. Lam, F.W., Wu, S.Y., Lin, S.J., Lin, C.C., Chen, Y.M., Wang, H.C., Chen, T.Y., Lin, H.T., Lin, J.H., 2011. The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae. Fish Shellfish Immunol. 30, 618–629. Langevin, C., van der Aa, L.M., Houel, A., Torhy, C., Briolat, V., Lunazzi, A., Harmache, A., Bremont, M., Levraud, J.P., Boudinot, P., 2013. Zebrafish ISG15 exerts a strong anti-viral activity against RNA and DNA viruses and regulates the interferon response. J. Virol. 87, 10025–10036. Lee, S.H., Peng, K.C., Lee, L.H., Pan, C.Y., Hour, A.L., Her, G.M., Hui, C.F., Chen, J.Y., 2013. Characterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle. Vet. Immunol. Immunopathol. 151, 217–228. Levraud, J.P., Boudinot, P., Colin, I., Benmansour, A., Peyrieras, N., Herbomel, P., Lutfalla, G., 2007. Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system. J. Immunol. 178, 4385–4394. Liu, M., Reimschuessel, R., Hassel, B.A., 2002. Molecular cloning of the fish interferon stimulated gene, 15 kDa (ISG15) orthologue: a ubiquitin-like gene induced by nephrotoxic damage. Gene. 298, 129–139. Lukacs, M.F., Harstad, H., Bakke, H.G., Beetz-Sargent, M., McKinnel, L., Lubieniecki, K.P., Koop, B.F., Grimholt, U., 2010. Comprehensive analysis of MHC class I genes from the U-, S-, and Z-lineages in Atlantic salmon. BMC Genomics 11, 154. Mattijssen, S., Pruijn, G.J., 2012. Viperin, a key player in the antiviral response. Microbes Infect. 14, 419–426. O’Farrell, C., Vaghefi, N., Cantonnet, M., Buteau, B., Boudinot, P., Benmansour, A., 2002. Survey of transcript expression in rainbow trout leukocytes reveals a major contribution of interferon-responsive genes in the early response to a rhabdovirus infection. J. Virol. 76, 8040–8049. Purcell, M.K., Garver, K.A., Conway, C., Elliott, D.G., Kurath, G., 2009. Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake Idaho. J. Fish Dis. 32, 619–631. Randall, R., Goodbourn, S., 2008. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J. Gen. Virol. 89, 1–27. Sun, B.J., Nie, P., 2004. Molecular cloning of the viperin gene and its promoter region from the mandarin fish Siniperca chuatsi. Vet. Immunol. Immunopathol. 101, 161–170. Verrier, E.R., Langevin, C., Benmansour, A., Boudinot, P., 2011. Early antiviral response and virus-induced genes in fish. Dev. Comp. Immunol. 35, 1204–1214. Yeh, Y.C., Lee, C.W., Pan, Y.W., Hsu, Y.J., Hung, H.Y., Chen, Y.M., Lin, H.Y., Chen, T.Y., Yang, H.L., Wang, H.C., 2012. Identification and characterization of DSCAM isoforms isolated from orange-spotted grouper Epinephelus coioides. Dev. Comp. Immunol. 38, 148–159. Zhang, Y.B., Jiang, J., Chen, Y.D., Zhu, R., Shi, Y., Zhang, Q.Y., Gui, J.F., 2007. The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells. Dev. Comp. Immunol. 31, 232–243.