Fish & Shellfish Immunology 31 (2011) 919e924
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Fish & Shellfish Immunology
journal homepage: www.elsevier.com/locate/fsi
Antigenic identification of virion structural proteins from infectious spleen
and kidney necrosis virus
Xiao-Peng Xionga, Chuan-Fu Donga, Shao-Ping Wenga, Jing Zhanga, Ye Zhanga, Jian-Guo Hea, b, *
a
b
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang West Road, Guangzhou 510275, People’s Republic of China
Key Laboratory of Aquatic Products Safety, The Ministry of Education, School of Marine Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2010
Received in revised form
5 August 2011
Accepted 19 August 2011
Available online 26 August 2011
Infectious spleen and kidney necrosis virus (ISKNV), belonging to the genus Megalocytivirus in the family
Iridoviridae, is one of the major agents causing mortality and economic losses to the freshwater fish
culture industry in Asian countries. Currently, little information regarding the antigenic properties of
Megalocytivirus (especially ISKNV) is available. Our previous study using four different workflows with
systematic and comprehensive proteomic approaches led to the identification of 38 ISKNV virionassociated proteins (J. Virol. 2869e2877, 2011). Thus, in this report, the antigenicity of 31 structural
proteins from ISKNV virion was investigated. A one-dimensional gel electrophoresis immunoblot profile
coupled with MALDI-TOF-TOF MS/MS was applied to identify six immunogenic viral proteins, namely,
ORFs major capsid protein (006L), 054L, 055L, 101L, 117L, and 125L. Then, the antigenicity of 31 structural
proteins was characterized by Western blot by using pooled sera from mandarin fish that survived ISKNV
infection. Of the 31 viral proteins, 22 were recognized by the fish ISKNV antiserum. Furthermore, this
antiserum neutralizes MFF-1 cells ISKNV infection. To our knowledge, this study is the first report on the
immunogenicity of viral proteins and characterization of the proteome of megalocytivirus infective
agents. Our findings are expected to promote the development of effective vaccine candidates.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Iridovirus
Megalocytivirus
ISKNV
Structural protein
Antigenicity
1. Introduction
In recent years, Iridoviruses are recognized as the major fish
pathogen in the marine aquaculture industry worldwide [1]. These
viruses are a group of large, cytoplasmic, icosahedral viruses with
a linear double-stranded DNA genome [2]. Iridoviruses are a family
of animal viruses that infect invertebrates (Iridovirus and Chloriridovirus) and poikilothermic vertebrates (Ranavirus, Lymphocystivirus, and Megalocytivirus) [3e5].
Infectious spleen and kidney necrosis virus (ISKNV) is the type
species of the genus Megalocytivirus, that cause severe mortality
and economic losses to the mandarin fish culture industry in China.
It has been increasingly recognized as a highly pathogenic virus
with commercial importance. Huge losses in aquaculture production due to this virus have been reported, especially in Asian
countries [6]. A recent epidemiologic investigation reported that
* Corresponding author. State Key Laboratory of Biocontrol, School of Life
Sciences, School of Marine Sciences, Sun Yat-sen University, 135 Xingang West
Road, Guangzhou 510275, People’s Republic of China. Tel.: þ86 20 84113793;
fax: þ86 20 84113229.
E-mail address: [email protected] (J.-G. He).
1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2011.08.009
ISKNV-like viruses can infect more than 50 types of cultured and
wild marine fish in the South China Sea [7]. Thus, intensive efforts
should be undertaken to prevent and limit the spread of megalocytiviruses in the fish industry.
Structural proteins, as the component of mature virion particles,
are of high interest in functional investigations of viruses because
they come into direct contact with host’s tissues or cells. They play
a crucial role in the course of viral infections and are often involved
with induction of immune responses and antigen recognition.
However, little information on the antigenicity of structural proteins
from ISKNV is available. Recently, several groups have successfully
applied immunoproteomics in systematically screening potential
immunogens from pathogens [8,9]. Studies have shown that the
structural protein of the virion-based vaccines have protected or
increased the survival rate of infected fish or shrimp [10,11]. Kim
et al. reported that survival from WSSV infection could be obtained
in shrimp by using WSSV structural proteins VP19 and VP466 as
a protein vaccine [12]. Hajime et al. identified 351R, a major capsid
protein of red sea bream iridovirus, as a potential vaccine against
Megalocytivirus infection in red sea bream [11]. With this in mind,
we performed a comprehensive analysis of ISKNV viral protein
antigenicity to promote the study of the pathogenesis of viral
infection and development of effective vaccine candidates.
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X.-P. Xiong et al. / Fish & Shellfish Immunology 31 (2011) 919e924
injected into the healthy mandarin fish. After 15 days postinfection, the sera of survivor fish were collected for Western
blotting analysis.
2. Materials and methods
2.1. Purification of ISKNV virions
ISKNV strain NH060831 purification was performed using the
double-sucrose density gradient method with minor modifications
[13]. Briefly, ISKNV-infected MFF-1 cell suspension was thawed
from storage at 80 C and was centrifuged at 6000g for 30 min at
4 C. The pooled supernate was centrifuged at 100,000g for
30 min at 4 C. The pellet was resuspended with PBS and ultrasonicated. The virus suspension was overlaided on 35% sucrose and
further pelleted by centrifugation at 150,000g for 1 h at 4 C to
remove cell debris. The resulting pellets were resuspended in TN
buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl) by gentle ultrasonic
treatment briefly. Then, the crude virus suspension was layered
onto a 30e60% sucrose gradient and centrifuged at 200,000g for
1 h 4 C. The milk-like pure viral band was collected and centrifuged at 100,000g for 30 min at 4 C. The pellet was resuspended
with PBS and then reloaded onto a sucrose gradient for ultracentrifugation. The virions were examined by negative staining under
transmission electron microscopy (TEM) to check for quality and
quantity.
2.2. Preparation of fish serum
The mandarin fish were maintained in well aerated dechlorinated water at temperature 25 C. Prior to viral infection, the
mandarin fish was transferred to the isolated flow-through tank
and allowed to acclimate for 10 days. For the infected mandarin fish
(n ¼ 40), 100 TCID50 purified ISKNV supplemented with 50 IU/mL
penicillin and 50 mg/mL streptomycin was intraperitoneally
2.3. SDS-PAGE and Western blotting analyses
SDS-PAGE of viral proteins was carried out using a 4% stacking
gel (pH 6.8) and 12% separating gel (pH 8.8) [14]. All samples were
heated for 5 min in boiling water and subjected to electrophoresis
at a constant voltage of 120 V until the dye front reached the
bottom of the gel. After separation, the SDS-PAGE gels were either
visualized by Coomassie brilliant blue-R250 (Sigma, USA) staining
or else transferred to nitrocellulose (NC) membranes in a transfer
buffer (48 mM Tris, 39 mM glycine, and 20% methanol) at 4 C. The
NC membranes were blocked with 5% skim milk in TNT buffer
(50 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.0) at 24 C for
1 h. After being rinsed three times for 10 min with TNT buffer, the
NC membranes were incubated with mandarin fish anti-ISKNV
serum (1:200) in a TNT buffer (containing 5% skim milk) for 1 h
at room temperature. Subsequently, the blot was rinsed three times
and incubated with secondary antibody (1:1000) rabbit anti-fish
conjugated with alkaline phosphatase for 1 h at room temperature on a gentle shaker. The membranes were then washed with
TNT buffer and the detection was performed with Nitro blue
tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP)
(Roche).
2.4. Protein identification by MALDI-TOF/TOF mass spectrometry
Mass spectrometric analysis was performed in accordance with
a previously described procedure [15]. Briefly, stained protein
Table 1
Recombinant proteins used in the present study.
No.
Protein namea
NCBI accession No.
Mrb (Da)
1-DE WB
Fish serum
Predicted structure and/or functionc
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
ORF
Q8V5D9
Q8QUV3
Q8QUU8
Q8QUU6
Q8QUT8
Q8QUS7
Q8QUS4
Q8QUS3
Q8QUS2
Q8QUS1
Q8QUR9
Q8QUR7
Q8QUR6
Q8QUR5
Q8QUQ6
Q8QUQ5
Q8QUQ4
Q8QUP7
Q8QUP2
Q8QUN9
Q8QUN4
Q8QUN2
Q8QUM9
Q8QUL7
Q8QUL5
Q8QUL0
Q8QUK9
Q8QUK1
Q8QUJ3
Q8QUJ2
Q8QUI6
49,866
51,704
12,940
35,075
54,732
36,952
38,866
50,275
53,460
32,298
49,399
13,337
30,071
34,670
34,919
34,583
23,482
101,661
53,835
61,253
112,965
17,199
18,550
32,701
42,805
20,044
19,182
104,713
52,503
50,160
25,160
þ
þ
þ
þ
þ
þ
þ
þ
Major capsid protein
Myristylated membrane protein, TM
Putative zinc finger protein
TM
TM
TM, SP
TM
RGD
Deoxyribonucleoside kinase
TM
TM
TM
006L
007L
012R
014R
022L
033L
036R
037L
038L
039R
041L
043L
044L
045L
054L
055L
056L
063L
068L
071L
076L
078R
081R
093L
095L
100L
101L
109L
117L
118L
125L
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Virus-specific 2-cysteine adaptor
Serineethreonine protein kinase, RGD
TM
Putative NTPase, RGD, TM
Hypothetical protein, TM
Hypothetical protein, TM
þ
þ
þ
þ
þ
þ
þ
þ
Hypothetical protein, TM
TM, ATP/GTP site motif A
TM
Putative ankyrin repeat protein, TM, RGD
, no, þ, yes.
a
Based on the genome of the ISKNV NH060831; The accession number is AF371960.
b
Predicted molecular mass based on the ORF.
c
Presence of putative structures are indicated. SP, signal peptides; RGD, cell attachment domain; TM, transmembrane domain.
X.-P. Xiong et al. / Fish & Shellfish Immunology 31 (2011) 919e924
921
were harvested by centrifugation at 10,000 rpm/min for 5 min,
then washed and resuspended in 50 mM sodium phosphate buffer
(pH 8.0) containing 8 M urea. Cell suspension was disrupted by
sonication in an ice bath (350 W, for 10 min 3 cycles), followed by
centrifugation at 7000g for 15 min at 4 C. The fusion proteins
were subsequently purified by affinity chromatography on Ni-NTA
Superflow resin according to the manufacturer’s instructions
(Qiagen, Germany). Protein concentrations were determined using
Bradford assay, and the protein samples were stored at 80 C until
use.
2.6. Neutralization assay
The neutralization assay was carried out with BD Falcontm
96-well cell culture plate. Briefly, MFF-1 cell were seeded in 96-well
tissue culture plates with monolayers were prepared in 96-well
tissue culture plates approximately 5 104 cells per well and
then cultured overnight at 25 C for forming cell monolayers. Prior
to inoculation, the anti-ISKNV fish antisera or normal (wild type)
fish antisera as control was treated at 56 C for 0.5 h. Then 50 ml of
a 1:10, 1:100 and 1:1000 diluted antisera were mixed with 50 ml of
ISKNV culture supernate. For neutralization assay, the virusantisera mixture was incubate at 25 C for 60 min. After incubation, ISKNV and antisera mixture was inoculated into each cell
monolayer of a 96-well cell culture plate. In the meantime, 50 ml of
103 pfu/ml of ISKNV culture supernate was also added into each well
as a positive control. The inoculums were removed from each well
after 1 h incubation at 25 C and 500 ml of DMEM culture medium
supplemented with 10% FBS was added to each well. On the 4th day,
a commercial cell counting kit CKK was used to analyze dead or live
virus-infected cells. The CKK-8 solution was added to each well.
After incubation for 1 h at 25 C, the absorbance of the mixture was
determined at 450 nm (reference, 630 nm) using MK3 (Thermo Lab
Systems), in duplicate.
Fig. 1. 1-DE Western blotting of viral proteins from ISKNV. Twenty micrograms of
structural proteins were separated by 12% SDS-PAGE. About 19 bands were detected
after staining with Coomassie brilliant blue. Of the 17 proteins, eight reacted with
ISKNV antibodies. Arrows indicate corresponding bands that were excised for MALDITOF/TOF MS/MS analysis. M: Molecular weight marker (kDa); (A) SDS-PAGE of purified
ISKNV virions; (B) Western blot using mandarin fish anti-ISKNV sera (1:200); (C) Intact
spherical ISKNV virions were observed by transmission electron microscopy. Scale
bar ¼ 100 nm.
bands were manually excised from the gel and washed twice with
50% acetonitrile in 25 mM ammonium bicarbonate for 15 min each
time. After they had been washed, the gel pieces were dehydrated
with 100% acetonitrile and then vacuum dried. The dried gel pieces
were rehydrated in 10 mM ammonium bicarbonate containing
0.1 mg/mL sequencing grade modified trypsin (Promega, USA) and
incubated at 37 C overnight. Trypsin-digested peptides were then
subjected to MALDI-TOF/TOF using the Applied Biosystems 4700
analyzer. The MS analysis was performed as previous description
[13]. MS peaks were selected between 800 and 3000 m/z. The
protein with the highest score (top rank), exceeding 95% confidence
level, was generally considered the candidate for the given protein.
3. Results
2.5. Purification of recombinant viral proteins
3.1. Virus purification
Thirty one recombinant plasmids of viral proteins from ISKNV
were available in our laboratory [13]. These were those codify by
ORFs 006L, 007L, 012R, 014R, 022L, 033L, 036R, 037L, 038L, 039R,
041L, 044L, 045L, 054L, 055L, 056L, 062L, 063L, 068L, 071L, 076L,
077R, 081R, 093L, 095L, 100L, 101L, 109L, 117L, 118L, and 125L
(Table 1). Purification of viral proteins was performed as previously
described, with a few modifications [16]. Overnight cultures of
Escherichia coli BL21 (DE3) harboring recombinant plasmids were
grown in fresh LB with ampicilin (100 mg/mL) from a 1:100 dilution
of starter inoculum, then incubated at 37 C until the optical cell
density reached about 0.6 of OD600. The bacterial culture was
induced with 1 mM IPTG (Sigma, USA) and allowed to grow for 3 h
at 37 C after the optimization of expression conditions. The cells
ISKNV virions were isolated from infected cells three days postinfection by sucrose gradient centrifugation. Sample purity is
crucial for analyzing the protein content of virions. The purity of
virion preparations was assessed by transmission electron
microscopy (TM) (Fig. 1C). TM analysis showed that the viral
purification was free of detectable cell debris and most of the
purified virions were intact.
3.2. Immunogenic analysis of proteins in ISKNV virions
After electrophoresis, gels were stained with Coomassie brilliant
blue or were transferred onto NC membranes for Western blot
Table 2
Identification of immunogenic viral proteins by 1-DE MALDI-TOF/TOF MS/MS.
Band Noa
Protein nameb
NCBI accession
No.
Mrc (kDa)
Coverage (%)
Score
Expect value
Queries
matched
Predicted structure and/or functiond
2
3
4
5
6
7
ORF
ORF
ORF
ORF
ORF
ORF
Q8QUJ3
Q8V5D9
Q8QUQ5
Q8QUI6
Q8QUQ6
Q8QUK9
53
50
35
25
35
19
35
21
44
54
40
24
179
88
206
151
86
71
5.2e-13
0.00064
1e-15
3.3e-10
0.0011
0.03
14
6
13
11
8
6
TM
Major capsid protein, TM
Serineethreonine protein kinase
Ankyrin repeat containing protein; TM; RGD
TM
No hit
a
b
c
d
117L
006L
055L
125L
054L
101L
Band numbers as shown in Fig. 1.
Based on the genome of the ISKNV NH060831; The accession number is AF371960.
Predicted molecular mass based on the ORF.
Presence of putative structures are indicated. SP, signal peptides; RGD, cell attachment domain; TM, transmembrane domain.
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X.-P. Xiong et al. / Fish & Shellfish Immunology 31 (2011) 919e924
Fig. 2. SDS-PAGE analysis of 31 recombinant proteins expressed in E. coli BL21 (DE3). M, molecular weight marker (kDa); P, purified protein; T, inducing total lysate protein.
analysis. SDS-PAGE result showed approximately 19 visible bands
ranging from 17 to 130 kDa with Coomassie blue staining (Fig. 1A).
Western blotting was used to detect immunogenic proteins on
1-DE using mandarin fish anti-ISKNV serum as the primary antibody. Eight bands were immunodetected (Fig. 1B), and were
excised from the gel. Following trypsin digestion of the reduced and
alkylated ISKNV proteins, the structural proteome of ISKNV was
analyzed by MALDI-TOF/TOF. Six of the detected proteins showed
peptide sequences that matched ISKNV ORFs (Table 2).
3.3. Expression and purification of recombinant structural proteins
Thirty one recombinant viral proteins (Table 1) were overexpressed and purify by affinity chromatography (Fig. 2). The
molecular masses of the recombinant proteins were well in
agreement with amino acid deduction. The molecular weights
obtained from SDS-PAGE analysis were in agreement with the
molecular masses deduced by amino acid sequences (Fig. 2).
043L, 044L, 054L, 055L, 063L, 076L, 095L, 100L, 101L, 109L, 117L,
118L, and 125L.
3.5. Virus neutralization assay
A neutralization assay was carried out by using the immune
antiserum from mandarin. In MFF-1 cells infected with a mixture of
ISKNV and normal (wild type) fish antisera, nearly all the infected
cells were dead three to four days post-infection (Fig. 4B). By
contrast, MFF-1 cells infected with the mixture of ISKNV and
antisera from mandarin fish anti-ISKNV were healthy, showing few
cytopathic effects, similar to the negative control (Fig. 4A and C).
Moreover, a CKK-8 assay was utilized to analyze dead or live virusinfected cells. The protective capacity of serum from survivor
mandarin fish was considerably higher than that of the control
(Fig. 4D). As above, the anti-ISKNV mandarin fish sera were able to
neutralize the cytotoxic activity of ISKNV on MFF-1 cells.
4. Discussion
3.4. Antigenicity of viral proteins
To investigate the immunogenicity of structural proteins, the 31
viral proteins above mentioned were quantified and transferred
onto NC membranes for Western blot analysis. The anti-ISKNV
serum from survivor mandarin fishs recognized 22 recombinant
proteins by Western blots (Fig. 3). The immuno-positive proteins
were ORFs 006L, 007L, 014R, 022L, 033L, 036R, 038L, 039R, 041L,
Megalocytiviruses have been isolated from more than 50 fish
species and are recognized as prevalent viruses that cause
considerable economic losses in the fish aquaculture industry
[17e19]. To date, few studies on antigen analysis of fish megalocytivirus have been conducted; essential to the functional analysis of these viruses is the identification and characterization of the
antigenicity of the viral proteins. In the present study, the antigenic
Fig. 3. Viral proteins recognized by Western blotting using mandarin fish anti-ISKNV serum.
X.-P. Xiong et al. / Fish & Shellfish Immunology 31 (2011) 919e924
923
Fig. 4. Neutralization assay in MFF-1 cell culture. (A) cells infected with ISKNV and fish anti-ISKNV serum; (B) cells infected with ISKNV and healthy fish serum; (C) cells infected by
medium as negative control; (D) CKK-8 assay on MFF-1 infected cells.
characterization of 31 ISKNV viral proteins was revealed. Furthermore, the six immunogenic proteins, which might be useful
for vaccine development, were identified by immnoproteomic
approach.
Many factors figure in the complexity of viral pathogenesis.
Understanding viral pathogenesis is crucial for developing vaccines
and treatments for viral diseases. One emerging area of research is
proteomics, the study of the viral protein component of the virions
[20e22]. Our earlier study using four different proteomic workflows led to the identification of 38 structural proteins, 3 of which
are envelop proteins [13]. Here, an attempt was made to screen
immunogen-related proteins from ISKNV particles by two different
strategies in parallel. First, using a combination of SDS-PAGE and
Western blotting assay, eight proteins were found by immunoproteomic methodologies and then identified by means of MALDITOF-TOF MS/MS. Six of these proteins were successfully identified
as 006L, 054L, 055L, 101L, 117L, and 125L. The major immunereactive band was identified as the major capsid protein
006L(MCP). The MCP is a well-known immunogenic protein in
other iridoviruses (insect iridoviruses [23], Singapore grouper iridovirus (SGIV) [24], and red sea bream iridovirus (RSIV) [25]), and it
has been used as immunogen in several vaccine trials [26]. 125L is
a vANK repeat protein, through interaction with the mandarin fish
IKKb shows its role in hibition of TNF-a-induced NF-kB signal
pathway [27]. Of the six immunogenic proteins, MCP is a known
immunogenic protein, five of which were first identified in this
study as immunogenic proteins. These immunogenic proteins
strongly reacted with fish blood after ISKNV challenge.
Western blotting was carried out with the use of the fish antiISKNV serum to detect and analysis the 31 viral antigens. In total,
22 recombinant structural proteins were recognized with the
presence of six viral antigens (i.e., ORFs 006L, 054L, 055L, 101L, 117L,
and 125L) also revealed by the combination of SDS-PAGE and
Western blotting (Table 2). In our previous study, 15 structural
proteins were revealed by the combination of 1-DE of the purified
virions and MS [13]. However, only 6 ISKNV proteins were revealed
by 1-DE of western blotting. It was difficult to reveal the lowabundance proteins by 1-DE, as well as the sensitivity of MS for
identification of protein [28]. Therefore we proposed another
strategy to further improve the limitation by analysis of quantitative recombinant protein, which provides more comprehensive and
precise antigen information. Of the newly recognized proteins,
previous report shown that five viral proteins (033L, 043L, 100L,
109L and 118L) were low-abundance proteins in the virion by
LC-MS [13], which could also trigger immune response; this
suggests that the immune response was not dependent on the
concentration of proteins in complex immunogens [9,29,30]. ORF
118L, a newly identified envelop protein, is homologous to a protein
encoded by ORF 112L in RBIV which displayed the ability of
antibody-neutralizing cytotoxic activity of RBIV [13,31]. ORF 007L is
one of another envelope proteins of ISKNV and is homologous to
the identified envelope protein ORF 53R of Rana grylio virus [32].
Recently, it reported that ORF 53R targeted artificial miRNAs that
could mediate iridovirus resistance to RGV infection by specific
silencing pathway [33]. The envelope protein is reported to be an
effective vaccine against WSSV [10,34]. There is a general agreement that anti-envelope antibodies that bind to the envelope spike
on the virion are neutralized or show antiviral activity [35]. The
usefulness of these identified envelop proteins to develop an ISKNV
vaccine is promising. Aside from the known 006L (MCP), ORF 125L
and 2 envelope proteins (ORF 007L and 118L), the immune function
of other 18 viral proteins in ISKNV virion is still fragmentary.
Taken together, the immunogenicity of the 31 structural
proteins was characterized by comprehensive analysis. This investigation of ISKNV antigenic properties and characterization of
infective agent proteomes will aid in understanding the characteristics of infection and pathogenesis of disease. Future work is
required for evaluation of immune functions of these identified
structural proteins.
Acknowledgments
This work was sponsored by grants from “863” project
(2006AA09Z445 and 2006AA100309), National Natural Science
Foundation of China (30325035 and U0631008).
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