MINISTRY OF EDUCATION

MINISTRY OF AGRICULTURAL

AND TRAINING

AND RURAL DEVELOPMENT

VIET NAM ACADEMY OF AGRICULTURAL SCIENCES
---------------

NGUYEN THI THANH

RESEARCH ON SOME BIOLOGICAL CHARACTERISTICS OF NERVOUS
NECROSIS VIRUS AND CREATION OF RECOMBINANT ANTIGENS USING
AS MATERIALS IN THE PRODUCTION OF VACCINES AGAINST DISEASES
IN GROUPERS (Epinephelus spp.)

SUMMARY OF AGRICULTURAL DOCTORAL THESIS

Ha Noi -2018


The study was completed at
VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

Supervisors
1. Ass.Prof.Dr. Pham Cong Hoat
2. Ass.Prof.Dr. Le Van Nam

Reviewer 1:...................................................................

preventive measures. Groupers are capable of stimulating an immune response when
exposed to antigens, therefore, it is necessary to research into the production of vaccines for
these species. Based on the practical needs we carried out the project:
" Research on some biological characteristics of nervous necrosis virus and
creation of recombinant antigens using as materials in the production of vaccines
against diseases in groupers (Epinephelus spp.)"
The research aims:
- To identifify the nervous necrosis virus (NNV) in groupers in Viet Nam perspectives and
their biological characteristics;
- To create recombinant antigens and evaluate their immunity stimulating capacity using as
materials in the production of vacinces against the VNN in groupers.
The scientific and practical contribution of thesis:
- The scientific contribution: The thesis identified some pathogenic viruses and some
of their biological characteristics. The thesis also produced the recombinant T4 protein of
the pathogenic virus, evaluated its capacity to stimulate immunity as a basis for the
production of vaccines against VNN in groupers.
- The scientific databases of the thesis will provide more materials for teaching and
researching on fish diseases and orientatiate the production of vaccines to against grouper
diseases.
- The practical contribution: The thesis created a recombinant antigen of T4 protein
and evaluated its ability to stimulate immunity using as materials to produce vaccines
against VNN in groupers. This will contribute to reduce infectious diseases, increase fish
production and sustainable development of grouper culture in general.

1


New contributions of thesis:
- This is the first thesis studying comprehensively on NNV in groupers in Vietnam. It
identified 26 viral strains and their biological characteristics.

1.2. Economic significance and current status of grouper culture
Groupers are of high economic value. For example, black and brown- spots
groupers with body weight between 800 and 1000g have been sold 200,000-300,000 VND/
kg, respectively. Red-spots groupers have prices ranging from 400,000 to 500,000 VND /
kg [73] [74]. Grouper aquaculture in Vietnam is mainly in coastal areas of Quang Ninh, Hai
Phong, Nghe An, Nha Trang, Ninh Thuan, Binh Thuan, Vung Tau and Kien Giang (Le Anh
Tuan, 2004 [11], Vo Van Quang et al, 2013 [5]). In recent years, the country has 500
hectares of coastal areas built into ponds for grouper farming. Annual production of
groupers reaches over 3000 tons of products. However, groupers are susceptible to several

2


diseases such as red spot, muscle necrosis, intestinal diseases which caused by bacteria and
NNV. Up to date, there is no vaccine to prevent these diseases and the main preventive
measure is to ensure hygiene within culture systems and isolate sources of viral infection
from fish.
1.3.
Current status of VNN in groupers in the World and Vietnam
1.3.1. Characteristics of VNN in marine fish
1.3.2. Current status of VNN in groupers in the World
1.3.3. Current status of VNN in groupers in Vietnam
1.3.4. Diagnostic methods for VNN in fish
Now a day, diagnostic methods used for detecting VNN are histopathology, viral
isolation in cells, molecular biology, electron microscopy and immunology (OIE, 2005)
[15].
1.4. Overview of nervous necrosis virus
The causative agents of VNN in groupers are NNV (belong to Betanodavirus),
which have RNA nucleus, spherical shapes and a diameter of 25-30 nm. Their genomes
have fragment structure, single-stranded RNA with two subdivisions. The large subspecies


diseases for grass carps, with the efficiency of 100% (Vu Dung Tien et al., 2003) [9]. Since
2003-2005, the KC-06-20NN project developed a vaccine against viscera necrotic
hemorrhage for pangasius and basa catfish. Vaccines are safe in experimental fish, with an
efficiency of 90% to 100% (Bui Quang Te et al., 2006).
Since 2006- 2007, the Research Institute for Aquaculture No.2 carried out a project
about research on the production of vaccines to prevent Edwardsiella ictaluri for pangasius
(Pangasianodon hypothalmus) comercially cultured in Cuu Long River Delta. The results
showed that the ability of immune response of pangasius to E. ictaluri bacteria through the
blood antibody is relatively high.
Since 2011, Pham Thi Tam et al. conducted research on the production of vaccines
against VNN in comercial farming goupers. The project outcome is the product of
inactivated vaccine by formalin 0.03% at 4°C for 5 days; the adjuvants is the ISA70
Montanique oil which is capable of stimulating the production of immune response in
laboratory conditions. Vaccines have been shown to protect groupers from diseases with an
efficiency over 83% in fingerlings, a 100% safe and an absolute asepsis (Pham Thi Tam et
al., 2015) [6].
CHAPTER 2. RESEARCH METHODOLOGY
2.1. Object and materials research
2.1.1. Object research:
- Nervous necrosis virus (NNV)
- T4 protein recombinant antigen
2.1.2. Materials research
- GS1 cell (Sigma, Germany)
- Vector pGEM-T and vector pET32a+ (Novagen, USA).
- E. coliJM109 and E. coliBL21(DE3) (HV Biotek).
- Enzyme EcoRI (Invitrogen), GS1
- RT-PCR one step kit (Quiagen), DNA Quick Gel Extraction kit (Invitrogen), DNA
and protein ladders (Invitrogen).
- Nikel chelating Resin column (Invitrogen)

- RT-PCR (Reverse Transcriptase PCR) technology.
- Electrophoresis on agarose gel: according to Sambrook et al., 2001.
- Gene isolation: according to Sambrook et al., 2001.
- Methods of creating T4 recombinant plasmid.
+ pGEM-T plasmid was cut by limited enzyme EcoRI, warm up at 37°C for 2.5 to 3
hours. Electrolytes on 1% agarose gel to test the results.
+ Linked T4 gene to pGEM-T:
- Methods to insert the recombinant vector into cells with host cell is E.coliJM109 by
temperature shock.
- Methods for extracting plasmid from E.coli bacteria:
- Methods for testing recombinant plasmid:
- DNA purification using agarose gel: according to the PureLink® Quick Gel
Extraction Kit included in the TOPO® TA Cloning Kit from Invitrogen.
- Sequencing of T4 gene by ABI 3100 automated machines (Applied Biosystems).
Blast software was used to determine the similarity of T4 gene sequences with GenBank.
2.3.2. Determination of some biological characteristic of grouper NNV
- Determine the virulence of virus NNV on cells: the virus was diluted from 10-1 to 109
with Leibovitz's 15 (10% FCS). The implanted tray was kept at 28oC to absorb the virus on
the cell. The virulence of NNV was assessed by TCID50.
- Determine the virulence of NNV in groupers: the virus was diluted from 10-1 to 10-9,
each one is injected in 30 groupers (1,5-2 cm) at a dose of 0.1 ml/fish. Clinical
manifestations, mortality and gene encoding T4 antigens were observed after 5 days. The
pathogenicity ability of the virus was assessed by LD50.
- Experiment on the effect of temperature on the ability of viral infection on GS1 cells:
QN4 strain with high TCID50 (10-6,8) was used to infect the cells. Each NNV strain was
cultured at 4 temperature levels: 17, 22, 27 and 32oC. The infection dose was TCID50 = 106,8
. Testing and evaluating of CPE were performed after 5 days of experiment.
- Experiment on the effect of temperature on the ability of viral infection in groupers:
QN4 strain with high LD50(10-7,5) was infected in groupers. Fish was injected with


(a) infected groupers, (b) vacuole in the brain, (c)
vacuole in the eyes.
Table 3.1. Detection of NNV by histopathology method
Cytopathogenicity in the brain tissue (26 Cytopathogenicity in the eyes tissue
samples)
(17 samples)
Sample ID
Ratio (%)
Sample ID
Ratio (%)

QN2, QN4, QN7
37,50
HP2, HP4, HP5, HP8, HP10
45,45
ND1, ND3, ND4, ND5,
72,72
ND7, ND8, ND10, ND11
KH4, KH5, KH6, KH9
44,44
BT1, BT2, BT4, BT7, BT9,
50,00
BT12
NNV was detected in the neuvous syndrome of

QN2, QN4, QN7
HP4, HP5, HP8
ND1, ND4, ND5,
ND10, ND11
KH4, KH5

33,33
58,33
52,94


The results show that 27/51 were positive with T4 gene, accounting for 52.94%. The PCR
products of HPV, HP2, ND3, ND2, QN2, QN4, KH4, KH5, BT2 and BT3 are shown in the
figure 3.2.
Samples of HP2, ND3,
QN2, QN4, KH4, KH5,
BT2, BT3 appeared to
have a band with a size of
approximately 420 bp,
which is similar to the size
of T4 gene. There was no
band on the samples of
HP1, ND2.
Hình 3.2. RT-PCR products of some samples infected with NNV on the

electrophoresic gel (well 1-10: HP1, HP2, ND3, ND2, QN2, QN4, KH4, KH5, BT2,
BT3, Well 11: marker)
3.1.2. Identification of NNV in cells
In this study, GS1 cell lines were used to assess the capacity of NNV infection. In
the total of 27 samples positive for T4 gene, we identified 26 virus strains (Figure 3.3, Table
3.3). HP11 did not show cytopathogenicity in the brain or eye tissues.

Figure 3.3. Cytopathogenicity of GS1 cells after NNV infection
A: The cells after 2 days NNV infection, granulocyte and rounded cells,
B: The cells after 7 days NNV infection,
C: Vacuolation in GS1 cells after 2 days NNV infection.

QN2
_
_
+
+
+
++
+++
2
QN4
_
+
+
+
++
+++
++++
3
HP2
_
_
+
+
+
++
+++
4
HP4
_
+

+++
++++
8
HP10
_
_
+
+
+
++
+++
9
HP11
_
_
_
_
_
_
_
10
ND1
_
+
+
+
++
+++
++++
11

+
+
+
++
+++
15
ND8
_
+
+
+
++
+++
++++
16
ND10
_
_
+
+
+
++
+++
17
ND11
_
_
+
+
+

BT2
_
_
+
+
+
++
++++
22
BT3
_
+
+
+
++
+++
++++
23
BT4
+
+
+
++
+++
++++
24
BT7
_
+
+

Therefore, they were thought NNV. In order to prove this hypothesis, we sequenced the T4
gene and determined them at the species level.
3.1.4. Sequencing T4 antigen- encoding genes of NNV
T4 gene of representative strains of QN2, HP7, ND1, KH5, BT12 were purified to
isolate and sequence. The results below show the steps of T4 gene sequencing of strain
KH5.

9


Figure 3.5. Design of the
pGEM-T-T4 recombinant
vector
A: Electrophoresis of the
purified T4 gene product
(well 1: purified T4 gene
product, M: 1kb Plus
Ladder well); B: Diagram
of T4 gene isolation vector
design
The purified product was approximately 420bp, which is similar to the size of T4
gene (Figure 3.5A). Splitting by pGEM-T Easy vector, recombinant vector were designed as
Figure 3.5B. Vector recombinant pGEM-T-T4 was transformed into E.coliJM109. White
colonies were selected and cultured in the liquid LB enviroment (ampicillin 100μg / ml) to
separate recombinant plasmids and cut, purify the T4 gene. Plasmid DNA was extracted
from the white colonies and tested on a 1% agarose gel and showed in th Figure 3.6 (A), the
T4 gene amplification product was examined and showed in the Figure 3.6 (B) the produce
was cut by the limited enzyme showed in the figure 3.6 (C).

Figure 3.6. Electrophoresis of recombinant T4 products

10-6,9
10-6,9

10-7
10-7

10-6,9
10-6,8

10-6,8
10-6,8

10-6,9
10-6,8

10-4,3
10-5
10-4,8

10-4,4
10-4,9
10-4,8

10-4,3
10-4,8
10-4,7

10-4,5
10-4,9
10-4,9


10-6,8
10-3
10-4,9

10-6,8
10-3,1
10-4,9

10-7
10-3
10-4,8

10-6,9
10-3
10-4,9

10-6,8
10-3
10-4,9

1
2

QN2

3
4
5



12

ND5

10-5,9

10-5,8

10-6

10-5,9

10-5,9

13

ND7
ND8

10-4,3

10-4,3

10-4,2

10-4,3

10-4,3


10-6,8
10-3

10-6,8
10-3

10-6,8
10-3,1

10-6,9
10-3

10-6,8
10-3

10-4,8

10-4,9

10-4,9

10-5

10-4,9

10-4,8
10-5,9

10-4,8
10-5,9

10-4,9

10-3,9
10-2,8
10-4,9

10-3,9
10-2,7
10-4,9

14
15
16
17
18
19
20
21
22
23
24
25
26

ND10
ND11
KH4
KH5
KH9
BT2

QN2
2
10-7,5
10-7,6
10-7,5
10-7,5
QN4
3
10-7,5
10-7,5
10-7,4
10-7,5
HP7
4
10-5,4
10-5,6
10-5,5
10-5,5
ND1
5
10-6,3
10-6,2
10-6,4
10-6,2
KH5
6
10-6,5
10-6,5
10-6,4
10-6,5


Figure 3.8. Servival of cells after NNV infection
The results presented in the figure 3.8 show that 22oC is the most suitable for virus
reproduction and GS1 cell infection.
3.2.4. Influence of temperature on pathogenicity of NNV in groupers
The pathogenicity of NNV at 28oC (day and night) is shown in the table 3.8: The
group of groupers infected with QN4 had a mortality rate of 82.1% after 3 days, 100% after
6 days . The mortality rate of control group was 10% after 15 days. It can be seen from the
table that the cause of fish death is QN4 of NNV with the participation of T4 gene.
Table 3.8. The pathogenicity of the NNV in groupers at 28°C of day and night
.Time
Mortality rate of groupers (%)
T4 gene determination
(day)
1st
2nd
3rd
Average
Control Treatments Control
0

0

0

0

0

0


6,6

+

-

9

6,6

12

10,0

15

10,0

-

Note: +: positive with T4 gene
-: Negative with T4 gene
The pathogenicity of NNV in groupers at temperature water of 28°C (daytime) and 24°C
(night time) was shown in the table 3.9.

13


Table 3.9. The pathogenicity of NNV in groupers at temperature water of 28°C

0
85,5
100

0
0
+
+
3,3
6,6
10,0
10,0
Note: +: positive with T4 gene
-: Negative with T4 gene
o
o
At 28 C (day) and 24 C (night), mortality rate of fish was 85.5% after 3 days and
100% after 6 days. In the control group, the mortality rate was 10% after 15 days. The
causive pathogen containing the T4 gene was the QN4 of NNV infection
3.3. Research on creating recombinant antigens using as materials to produce vaccine
against VNN for groupers
3.3.3. pET32a+- T4 recombinant vector design.
The study used the pET32a+ vector to
express T4 gene, E. coliJM109 strain
to test recombinant vector and E.
coliBL21(DE3) strain to express T4
gene.
The process of creating recombinant
vector containing T4 gene (pET32a+T4) is shown in the figure 3.9.
pGEM-T-T4 and pET32a+ plasmid

vector
Well 1: T4 gene after purification, Well 2: pET32a+ plasmid after purification
M: 1kb Plus Ladder
Gene linked reaction was performed at 4oC and incubated for 14 to 16h. To test the
formation of the pET32a+-T4 recombinant vector, the gene-linking product was transformed
into E. coliJM109. Bacteria carry the recombinant vector on LB environment supplemented
with Ampicillin (100μg / ml) were screened and cultured at 37oC for 16h, results shown the
Figure 3.12. Six colonies were chosen and cultured in 3 ml of liquid LB environment
supplemented with ampicillin (100μg/ ml) and shaked with a speed of 150 times/minute at
37oC for 16h. The plasmid was then isolated, checked by electrophoresis on 1% agarose gel
and the result is shown in the figure 3.13:

15


Figure 3.12. a colony plate after transfering
Figure 3.13. Cheking the formation of
+
pET32a -T4 gene into E. coliJM109
pET32a+-T4 vector in E. coliJM109 (Well 1-6:
plasmid lines 1 to 6; Well M: 1kb plus ladder)
To test six plasmid samples which
carry the pET32a+- T4 recombinant
vector and pET32a+ vector
(control), a PCR reaction was run
using the pair of primer P1 and R3.
The results in the figure 3.14 show
that six selected bacterial strains
carried T4 gene, while the control
samples did not carry the gene.


To test the transformation ability of pET32a+-T4 plasmid into the E. coliBL21(DE3), a
sceening was performed by isolating plasmid then running PCR. The isolated plasmid
product was checked by electrophoresis on 1% agarose gel, the results are shown in the
figure 3.16

Figure 3.16. Electrophoresis of the
Figure 3.17. Checking the transformation ability
recombinant plasmid isolated from E.
of the pET32a+-T4 recombinant vector into E.
coliBL21(DE3) strain carry pET32a+-T4
coliBL21(DE3) strain.
vector
Well 1-4: Plasmid PCR products
Well 1-4: Plasmids;
Well M: GeneRulerTM 1kb DNA Ladder
Well M: GeneRulerTM 1kb DNA Ladder
A PCR reaction was perfomed to test plasmids’ capacity carying pET32a+-T4
vector. The results of electrophoresis on 1% agarose gel in the figure 3.17 show that there
was a band with a similar size of T4 gene, 420 bp on the wells 1 to 4. It is therefore possible
to conclude the E.coliBL21(DE3) strain carrying T4 gene of NNV has been successfully
created.
3.3.2.2. Expression of T4 gene in recombinant bacteria cells
Four selected colonies were innoculated until the OD600nm reaching 0.5 to 0.6, then IPTG
was added. The recombinant bacteria were sampled before and after adding IPTG 2h, 4h, 6h
for expression analysis of the antigen-encoding gene by SDS-PAGE. The results are shown
in the figure 3.18 and figure 3.19.

17


confirmed by Western blot
hybridization method with
specific antibody againsts
NNV. The results show
that the reaction site
between T4 recombinant
protein
and
standard
specific antibody was at
the 25 kDa electrophoresis
line (Figure 2.20). It is
therefore can be concluded
that T4 protein antigen of Figure 2.20. Western blot result of recombinant T4 protein
NNV was successfully
M: standard; Well 1: T4 recombinant protein; Well 2:
expressed
in
E.
standard T4 protein of USA
coliBL21(DE3) cells.

18


3.3.2.3. Assessment of expression conditions of T4 antigen-encoding gene of NNV
- Determination of sample collection time
The E. coli cells
carrying recombinant
plasmids were cultured


19


- Determination of the concentration of IPTG induction
IPTG was added to the
culture
medium
at
concentrations from 1 to
6 mM. The results in the
figure 3.23 show that,
after IPTG addition, the
recombinant
strain
synthesized
a
large
amount
of
protein,
approximately 25 kDa
(the theoritical size of the
recombinant T4 protein).
In experiments using
different
IPTG Figure 3.23. Effect of IPTG concentrations on recombinant
concentrations, the T4 T4 protein expression
Well M: standard, wells 1-6: IPTG concentrations from 1
gene was best expressed

Time to collect
serum in rabit
after
immunization
(day)

Positive
control

(CPE %)

(CPE %)

E.coli BL21- Recombinant
pET32a+-T4* T4 protein*

5

98

0

15

98

25

Negative control


1: 50

35

98

0

0

1: 800

1: 200

45

98

0

0

1: 800

1: 200

60

98


Infection Death
(day)
(%)
(%)

Negative control
(infection rates
%)

Serum rabbit sera were immunized

E.coli
recombinant T4
E.coli
Recombin
+
BL21- pET32a -T4
protein
BL21ant T4
pET32a+-T4 protein Neutralization T4 Neutralizati T4 gene
titer
gene
on titer

5

100

100


25

100

100

0

0

1: 100

-

1: 50

-

35

100

100

0

0

1: 100


0

0

1: 50

+

1: 10

+

(Note: ND: not done; (+): have T4 gene, (-): have T4 gene)
Table 3.11 shows that in both two experiments of rabbit serum immunized with E.
coliBL21-pET32a+-T4 and recombinant T4 protein, the serum did not produce antibody in
the first 15 days. On 25th to 45th days, only the rabbit serum immunized with E. coliBL21pET32a+-T4 had an ability to produce a neutralizing antibody against NNV virulence at a
dilution of 1: 100. From 25th to 35th days, the T4 antigen-encoding gene was not detected.
On 45th day, although a neutralization titer of the serum was found at a dilution of 1:50, all
groupers were not killed and T4 gene was detected. Although rabbit serum still produced
antibodies to neutralize virulence, the virus was no longer able to cause diseases in two
experiments, the neutralization titers ranged from 1:10 to 1:50 and the T4 gene was still
detected in experimental fish.
Above findings indicate that recombinant the T4 protein is abble to produce
antibody that neutralize pathogenic viruses, although the antibody is lower than that of the
E. coliBL21-pET32a+-T4.
3.3.4.2. Evaluation of ability to create antibodies of recombinant antigens in
groupers
The results presented in the table 3.12 show that in negative control samples, fish
immunized with E. coliBL21-pET32a+-T4 antigen and recombinant T4 protein produced a
specific antibody of NNV and did not cause cytopathogenic effects.

The fish extracted fluid was
immunizated with recombinant T4 protein
The fish extracted fluid was
immunizated with E.
1:64
1:256
1:128
1:128
1:64
1:8
coliBL21-pET32a+ -T4+ NNV
virulence
The fish extracted fluid was
immunizated with recombinant 1:16
1:128
1:128
1:64
1:64
1:8
T4 protein + NNV virulence
(Note: ND: not done; (+): have CPE, (-): no CPE)
In the positive control samples, NNV QN2 strain 104 TCID50 dose was infected into GS1
cells and all cells had cytopathogenic effects.
In both experiments, fish extracted fluid immunizated with E. coliBL21-pET32a+T4 and recombinant T4 protein produced antibodies from 15th day with titers of 1:64 and
1:16, respectively. In the experiment of grouper extracted fluid immunizated with the E.
coliBL21-pET32a+-T4, the antibody concentration was highest on 30th to 60th days with
titers of 1:128 -1:256. By 75th day, the antibody concentrations were decreased with a titer
of 1:64. In the experiment of grouper extracted fluid immunizated with T4 recombinant
protein, the antibody was highest on 30th to 45th day with a titer of 1:128. From 60th to 75th
day, the antibody was gradually decreaed and the titer was only 1:64. The protection lasted