Journal of Fish Diseases 2014, 37, 319–325
12105
Construction of KHV-CJ ORF25 DNA vaccine and immune
challenge test
J-X Zhou†, H Wang†, X-W Li, X Zhu, W-L Lu and D-M Zhang
Animal Product Quality & Safety Key Laboratory of the Ministry of Education, College of Animal Sciences,
Jilin Agricultural University, Changchun, China
Abstract
KHV ORF25 fragments were cloned from Koi
Herpes Virus-CJ (KHV-CJ) strains isolated in our
laboratory. The amplified products were inserted
into the eukaryotic expression vector pIRES-neo,
forming recombinant plasmid pIRES-ORF25.
The recombinant plasmid pIRES-ORF25 at 1 lg
per koi, 10 lg per koi and 50 lg per koi was
intramuscularly injected into healthy kois, respec-
tively. The results showed that the recombinant
pIRES-ORF25 could induce the production of
specific antibodies in koi determined by indirect
ELISA. The differences of immune effect between
three doses were not significant (P > 0.05), but
all of them could induce the production of neu-
tralizing antibodies. The immune challenge test
showed that the mortality of koi injected with
PBS, blank pIRES-neo vector and nothing was
90%, 92.5% and 85% at 25 days. While the
mortalities of koi injected with eukaryotic expres-
sion plasmid pIRES-ORF25 were 20%, 17.5%
and 12.5%. Differences in comparison with the
control group were highly significant (P < 0.01).
Histopathological staining revealed that the tissues
of the immunized koi did not change apparently.
In conclusion, the DNA vaccine pIRES-ORF25
construct could well protect koi against KHV and
had the potential to be applied in practice.
Keywords: immune challenge test, immunization,
koi, koi herpes virus, ORF25 DNA vaccine.
Correspondence D-M Zhang, Animal Product Quality & Safety
Key Laboratory of the Ministry of Education, College of Animal
Sciences, Jilin Agricultural University, Changchun 130118,
China (email: dmzhang@jlau.edu.cn)
†
These authors contributed equally to the work.
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doi:10.1111/jfd.
Introduction
Koi herpes virus disease (KHVD) is a highly con-
tagious and lethal diseases caused by the Kio her-
pes virus (KHV) in koi, carp and common
variants. In 1998, the disease first broke out in
Israel Maagan Michael and the United States
(Hedrick et al. 2000). In 2000, the disease spread
to the United Kingdom, Germany and Belgium
(Haenen et al. 2004). In April 2002, this disease
occurred in Indonesia, meanwhile Koi was sus-
pected for occurrence of the disease in Guangdong
China (Liu et al. 2002). In December 2002, Koi
in Taiwan, China, was confirmed to be infected
with KHV (Shih et al. 2007). In October 2003,
the disease was found in Japan (Iida & Sano
2005). Higher incidence of the disease in breeding
koi in China resulted in mass mortality of koi,
which almost destroyed the koi farming industry.
Currently there is no specific therapy for the dis-
ease. Vaccination is considered the primary means
of prevention but the current commercial vaccines
are all of doubtful effectiveness. Although attenu-
ated vaccine has a better effect, there is a risk of
spreading the virus and reacquiring its toxicity
(Perelberg et al. 2008). Compared with the atten-
uated vaccine, inactivated vaccine (Perelberg et al.
2005; Yasumoto et al. 2006) displays superior
security. Its weaker protective capacity, however,
and its instability, require repeated immunization.
It can be said that the lack of safe and effective
vaccine is the main obstacle in the prevention and
control of KHVD.
DNA vaccine is produced by inserting the pro-
tective antigen gene into the eukaryotic expression
vector using recombinant DNA technology. The
recombinant DNA is directly inoculated into the
body for endogenous antigen expression, which is
 Journal of Fish Diseases 2014, 37, 319–325 J-X Zhou et al. KHV-CJ ORF25 DNA vaccine immune challenge test
presented to the immune system for inducing the
body to produce specific humoral and cellular
immune responses (Woiff et al. 1990). Koi herpes
virus is a linear double-stranded DNA virus with
a genome size of 295 kbp. In fish herpes virus, it
has the largest genome. The research of the
whole-genome structure and its encoded protein
function is still in infancy and most of the protein
functions are unclear (Pikarsky et al. 2004). The
ORF25 gene encodes glycosylated proteins with
the cleavage site of signal peptide, and the expres-
sion is mainly present in the cytoplasm. The
ORF25 gene is the gene encoding the major
structural protein of KHV. After a series of bioin-
formation analysis, we forecast its good
immunogenicity.
In this study, we used the ORF25 gene as the tar-
get gene, pIRES-neo as eukaryotic expression vec-
tor, to construct eukaryotic expression recombinant
plasmid pIRES-ORF25. The immune validity of
this expression system was investigated for develop-
ing a new efficient DNA vaccine against KHV.
Materials and methods
Materials and reagents
China Jilin strain of koi herpes virus-CJ (KHV-CJ)
was isolated by our laboratory (Zhu et al. 2011).
Box mirror koi caudal fin primary cells (MFC)
were preserved in our laboratory. The eukaryotic
expression vector pIRES1-neo was donated by Vet-
erinary Institute of PLA, Academy of Military
Medical Sciences. Healthy koi Cyprinus carpio
Linnaeus that were shown to be negative for KHV
by PCR detection, with body weight of
200–300 g, were provided by Changchun Acad-
emy of Fishery Sciences, Jilin Province, China.
Restriction enzymes, Ex Taq DNA polymerase,
dNTP, pMD18-T vector, T4 DNA ligase and
DNA marker were purchased from Takara Bio-
technology Co., Ltd. Newborn calf serum and
M199 medium were purchased from Invitrogen.
DNA Gel Extraction Kit was from Tiangen Bio-
chemical Co., Ltd. Western blot prestained pro-
tein marker was from Beijing Biofuture Institute
of Bioscience & Biotechnology Development
(China). KHV-positive serum and horseradish
peroxidase enzyme-labelled rabbit anti-fish anti-
body were kept in our laboratory. Transfection
reagents FuGENE HD were purchased from
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Roche. Other reagents were homemade analyti-
cally pure products.
Construction and characterization of
eukaryotic expression plasmid pIRES-ORF25
Referenced to KHV ORF25 gene sequences in
GenBank, a pair of primers was designed using
primer design software PRIMER PREMIERS 5.0
(Shanghai Biological Technology Services Ltd.)
The sequences were as follows: P1 5′-GAT-
ATCATGTCATGTTCCATCCGCCA-3′ (under-
lined parts for the sequence of EcoRV);
P2 5′-GAATTCTTAGGTGGCGTTGAGGTC-3′
(underlined parts for the sequence of EcoRI).
Using KHV-CJ strains as a template, PCR ampli-
fication was performed. PCR reaction conditions
were as follows: predenaturation at 95 °C for
5 min; denaturation at 94 °C for 40 s, annealing
at 59.3 °C for 30 s, extension at 72 °C for
1 min, 35 cycles; final extension at 72 °C for
10 min, kept at 4 °C. PCR products were sepa-
rated on 1% agarose gel and recycled according to
nucleic acid extraction kit instructions. The PCR
products ligated with pMD18-T vector and trans-
formed into Escherichia coli, followed by plasmid
extraction and analysis by enzyme digestion.
The extract plasmids and pIRES-neo vector
underwent double digestion, respectively, under
the following conditions: 1 lL of EcoRVand
1 lL of EcoRI, 2 lL of 10 9 H buffer, 10 lL of
pMD18-T-ORF25 plasmid or 10 lL of pIRES-
neo vector, 6 lL of ddH2O, total volume of
20 lL. After separation on gel, ORF25 fragment
and the large fragment of the pIRES-neo vector
were recycled, respectively. They were ligated at
16 °C overnight under the reaction system as fol-
lows: 5 lL of ORF25 fragment, 1 lL of large
fragment of the pIRES-neo vector, 1 lL of
10 9 T4 buffer, 1 lL of T4 DNA ligase, 2 lL
of ddH2O, total 10 lL. Ligation product was
transformed into E. coli DH5a, and positive
clones were selected and proganated, followed by
a small-scale extraction of plasmid and identifica-
tion using restriction enzyme. Positive bacteria
were used for massive plasmid preparation. MFC
cells were transfected with the constructed eukary-
otic expression plasmid pIRES-ORF25 according
to steps in the FuGENE HD kit (Roche). After
36 h, cells were collected, followed by Western
blotting analysis.
 Journal of Fish Diseases 2014, 37, 319–325
Immunizing animals in groups
A total of 240 healthy koi with the body mass of
200–300 g were randomly divided into six groups
(n = 40 in each group). The aquarium (volume
0.8 m3) temperature was at 25  1 °C. Koi in
Group 1 was injected with PBS, in Group 2 with
blank pIRES-neo vector, in Group 3 with nothing
and in Groups 4, 5, 6 with the recombinant plas-
mid pIRES-ORF25 at a dose of 1, 10, and
50 lg, respectively. Immunization volume was
1 mL. The concentration of plasmid was adjusted
by physiological saline solution . After the first
immunization, the second and third immuniza-
tions were performed every 3 weeks. Five ran-
domly selected koi underwent sterile blood
collection by heart before immunization and every
week after immunization, until two weeks after
the third immunization. The blood was placed at
37 °C for 1 h and 4 °C for 2 h, followed by
centrifugation at 400 g for 15 min to separate
serum, which was stored at 20 °C for the detec-
tion of antibody.
Detection of antibody levels in koi serum by
ELISA
The KHV isolated by our laboratory was 100-fold
diluted with carbonate buffer (pH 9.6, TCID50 =
10 6.75) and added to 96-well microtitre plates,
100 lL in each well, coating at 4 °C overnight.
The consecutive procedures were performed. The
liquid was discarded, adding 5% skim milk in
PBS 37 °C for 1 h. Each well was filled by
100 lL of the 10-fold diluted koi serum at 37 °C
for 1 h. Both positive and negative control wells
were treated as the same procedures. Each well was
filled by 100 lL of horseradish peroxidase-labelled
rabbit anti-fish IgM diluted with PBS at 37 °C
for 1 h. After each step, the liquid was discarded,
and the plate was washed with PBST for three
times, 3 min for each time, and drying plates on
absorbent paper. Each well was filled by 100 lL
of tetramethylbenzidine (TMB) substrate in darkness
for 5–15 min, then stopping the reaction with 2 M
H2SO4). The absorbance (A) of each well was
measured at 450 nm.
Detection of antibody titre by neutralization tests
The serum dilution method was used to deter-
mine the anti-KHV neutralized antibody titre in
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J-X Zhou et al. KHV-CJ ORF25 DNA vaccine immune challenge test
koi serum after immunization by the recombinant
plasmid. The koi serum was diluted at ratios 1:2
to 1:128 with two-fold dilution and mixed with
equal volume of KHV containing 100 TCID50
(TCID50 = 10 6.75) at 37 °C for 1 h. The mix-
ture was added to cell culture plate, four replicates
for each sample, 100 lL per well. Adding 100 lL
to caudal fin cell (MFC) suspension to each well,
the plate was incubated at 27 °C in 5% CO2,
controlled by positive serum, negative serum,
virus, healthy cells without addition. The disap-
pearance of lesions in each well was regarded as
positive criteria, and its highest dilution titre was
set as the neutralizing end, calculating serum
neutralizing titre according to the Reed-Muench
formula (Cunningham 1973).
Immune challenge test and protective
percentage
Two weeks after the third immunization, the
aquarium (volume 0.8 m3) temperature was at
25  1 °C; immune challenge test was per-
formed by inoculating KHV-CJ (TCID50 =
10 6.75) by thoracic cavity, followed by recording
the morbidity and mortality. As koi in the control
group began to die, two koi in each group were
randomly selected and killed to collect their bran-
chia, kidney, liver and spleen. Tissues were fixed
with 0.4% neutral formalin and then paraffin sec-
tions were prepared to observe the pathological
changes.
Data analysis
All data were analysed using software SPSS 13.0.
(WuHan Sinfei Information Technology Co.
Ltd.) P < 0.05 indicated significant differences,
and P < 0.01 extremely significant differences.
Results
Detection of recombinant plasmids and
expression
PCR amplified 849 bp from KHV-CJ template,
consistent with the expected (Fig. 1). Double
digestion by EcoRV and EcoRI produced 2860
and 849 bp fragments (Fig. 2), consistent with
the expected, indicating that the ORF25 gene
was successfully cloned into the pMD18T vector.
The double digestion of the extracted
 Journal of Fish Diseases 2014, 37, 319–325 J-X Zhou et al. KHV-CJ ORF25 DNA vaccine immune challenge test

M 1 M 1
8000

2000 5000
5237 bp
3000
1000
849 bp 1500
750
1000
500 849 bp
500
250
100
Figure 3 Identification of recombinant plasmid pIRES-
ORF25 digested by EcoRV and EcoRI. M: 8000DNA Marker;
1: Results of recombinant plasmid by double digestion.
Figure 1 PCR amplification of Kio herpesvirus China Jilin
strain ORF25 gene. M: DL2000DNA Marker; 1: PCR prod-
uct of ORF25 fragment.
kD M 1 2
97.4
M 1 66.2
8000
43.0
5000
3000
31.0
1500

1000 849 bp 20.1

500 Figure 4 Analysis of protein by Western blotting. M: Marker;

1: pIRES-ORF25 group; 2: negative control.

Figure 2 Double digestion for pMD18T-ORF25. M:

8000DNA Marker; 1: product from pMD18T-ORF25
digested by EcoRV and EcoRI.
recombinant pIRES-ORF25 produced 5237 and
849 bp fragments, which were in accordance with
the length of pIRES-neo vector digested by
EcoRV and EcoRI and ORF25 fragment, respec-
tively (Fig. 3), indicating that ORF25 gene was
inserted to pIRES-neo vector. MFC cells were
transfected with eukaryotic expression plasmid
pIRES-ORF25; 36 h after transfection, cells were
collected and detected by Western blot analysis.
Specific reaction band (32 kD) was showed,
while no specific bands (Fig. 4) were showed
in cells transfected with pIRES-neo vector. This
result confirmed that eukaryotic cells could
express target gene carried in the recombinant
plasmids.
Detection of antibody levels in the serum of
immunized koi
Serum separated at different time was detected
using indirect ELISA. The average of antibody
levels was obtained in each group, plotting a curve
of koi serum antibody levels (Fig. 5). It can be
seen that compared with Group 1 (PBS), Group
2 (pIRES-neo vector) and Group 3 (blank con-
trol), one week after the first vaccination, antibody
levels in Group 4 (pIRES-ORF25 1 lg), Group 5
(pIRES-ORF25 10 lg) and Group 6 (pIRES-
ORF25 50 lg) significantly increased (P < 0.01).
After the second and third immunization, anti-
body levels continued to rise (P < 0.01). Two
weeks after the third immunization, antibody lev-
els were basically at the peak; differences of anti-
body levels between Groups 4, 5,6 were not
significant (P > 0.05).

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 Journal of Fish Diseases 2014, 37, 319–325 J-X Zhou et al. KHV-CJ ORF25 DNA vaccine immune challenge test

Figure 5 Specific IgG responses of koi

immunized with recombinant plasmid
pIRES-ORF25. Each group of koi
(n = 40) was immunized i.m. with
different doses (1 lg, 10 lg, 50 lg) of
DNA at 0, 3 and 6 weeks. Time (week)

Detection of neutralizing antibody levels Pathological section in immune challenge test

The neutralizing experiment for the detection of
antibody titre showed that no antibody was pro-
duced in the group with intramuscular injection
of PBS, blank vector and nothing; the neutralizing
antibodies were produced in the group immunized
by recombinant plasmid (Table 1).
Immune challenge test and the protective effect
Two weeks after third immunization, immune
challenge test was performed by injection of
KHV-CJ with 100TCID50 (TCID50 = 10 6.75).
Eight days after inoculation, fish began to die,
mortality continued for 20 days, when it ceased.
Statistical results (Table 2) showed that the mor-
tality rate for koi injected with PBS, empty
pIRES-neo vector and nothing was 36, 37 and
34, the mortality rates 90%, 92.5% and 85%.
The number of fish dying in treatment group
injected with three concentrations of eukaryotic
expression plasmid pIRES-ORF25 was eight,
seven and five, the mortality rates 20%, 17.5%
and 12.5%. These results showed that the con-
structed DNA vaccine – pIRES-ORF25 – had a
better protective effect against KHV.
Microscope sections stained with H&E were exam-
ined at 100X magnification. Two biopsies of koi
injected with pIRES-neo vector were observed. Fig-
ure 6a shows one of them. The gill structure was
degenerate, there was interstitial inflammation and
hyperaemia accompanied by interstitial cell necro-
sis, slight splenic haemorrhage and cytolytic necro-
sis of spleen and liver. The biopsy of koi injected
with PBS and nothing showed the histological
appearance similar to the blank vector’s (Figure not
shown). The biopsy of koi injected with a series of
concentrations of pIRES-ORF25 showed normal
histological appearance (Fig. 6b–d). There was no
marked difference in histological appearance
between koi injected with three concentration of
pIRES-ORF25.
Discussion
KHV is now widespread. (Sano et al. 2004;
Neukirch & Kunz 2001; Oh et al. 2001.) It has
since its first detection caused major economic
losses in China but inactivated virus has proved
disappointing in stemming its progress. Our
experimental results described in this paper

Table 1 Detection of antibody against KHV in koi by neutralization test

Group PBS pIRES-neo Blank pIRES-ORF25(1 lg) pIRES-ORF25 (10 lg) pIRES-ORF25 (50 lg)

Before immunization <1:2 <1:2 <1:2 <1:2 <1:2 <1:2

1 week after 1stimmunization <1:2 <1:2 <1:2 <1:4 <1:4 <1:4
2 weeks after 1st immunization <1:2 <1:2 <1:2 <1:8 <1:8 <1:32
1 week after 2nd immunization <1:2 <1:2 <1:2 <1:32 <1:8 <1:32
2 weeks after 2nd immunization <1:2 <1:2 <1:2 <1:32 <1:32 <1:64
1 week after 3rd immunization <1:2 <1:2 <1:2 <1:64 <1:64 <1:64
2 weeks after 3rd immunization <1:2 <1:2 <1:2 <1:64 <1:64 <1:128

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 Journal of Fish Diseases 2014, 37, 319–325 J-X Zhou et al. KHV-CJ ORF25 DNA vaccine immune challenge test

Table 2 The result of death rate after inoculation the recombinant plasmid by fish, resulting in dif-
pIRES-ORF25
ferent immune effects. Some studies have shown
that intramuscular injection of DNA vaccine dis-
Group PBS pIRES Blank (1 lg) (10 lg) (50 lg) plays a better immune effect for its inducing the
Total 40 40 40 40 40 40 expression of protective antigen (Gomez-Chiarri
number et al. 1996), which will provide fish with a strong
Death 36 37 34 8 7 5
and long-lasting immunity. In the present study,
Death 90 92.5 85 20 17.5 12.5
rate(%) the most commonly used intramuscular injection
route was used to immunize koi separately with
recombinant pIRES-ORF25, which resulted in a
indicate that recombinant pIRES-ORF25 can good response.
induce high levels of antibody and good For traditional inactivated vaccine and
protection. attenuated vaccine, performing an enhanced
Currently, vaccination approaches of DNA vac- immunization after the first immunization will
cine for fish generally include intramuscular injec- substantially boost the protective immunity (Jansson
tion, oral administration, subcutaneous injection & Ljungberg 1998). In the present study,
and soaking. Using different ways of vaccination twice-enhanced immunizations following a basic
will directly affect the distribution and uptake of immunization did strengthen the immunity, dem-
onstrating an increased antibody levels by the

Branchia Kidney Spleen Liver

(a)

(b)

(c)

(d)

Figure 6 (a) Microscopic histological staining of tissues from koi injected with pIRES-neo vector, (b,c,d) 1 lg,10 lg and 50 lg of
pIRES-25 (9100 magnification).

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 Journal of Fish Diseases 2014, 37, 319–325
enhanced immunization. A series of biosafety trials
above are going to be performed so that it gets a
truly safe and effective DNA vaccine.
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Received: 3 November 2012
Revision received: 25 February 2013
Accepted: 25 February 2013