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 Ass.Prof.Dr Pham Cong Hoat Ass.Prof.Dr Le Van Nam Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended at the Institute Council in meeting room of Vietnam Acadermy of Agricultural Sciences, at: hr date month year 2018 The dissertation is available in Libraries : National Library of Vietnam Vietnam Acadermy or Agricultural Sciences Instutute for Agricultural Genetic INTRODUCTION In recent years, fisheries have been growing rapidly and become one of the important economic sectors of Vietnam Marine fish farming is evaluated as a high economic efficiency of aquaculture and the grouper is considered one of the key species Groupers (Epinephelus spp.) have a high economic value because of high nutrient contents, delicious meat, thus they are popular in both dometic and international markets However, when grouper farming grows, farmers face many difficulties because of fish diseases Several studies have shown that major causive pathogens of groupers are viruses, fungi and bacteria Of those, the most serious one is Betanodavirus, which causes viral nervous necrosis (VNN) or encephalopathy (Viral Encephalopathy and RetinopathyVER) The Betanodavirus can cause VNN in groupers at all developmental stages from larvae, fingerlings to commercial sizes Common symtoms of infected fish are manifestations of nervous system disorders, which can be seen as fish swimming unbalanced, spinning, swimming upside down or hanging on water surface or at the bottom of culture tanks and cages Infected fish can be died after 3-5 days with a high mortality rate of 80-100% (Do Thi Hoa et al., 2004) The outbreak of the VNN in groupers has increasing and awaisting effectively 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 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 - This is also the first research in Vietnam to successfully create the recombinant antigen, T4 protein, which can stimulate immune responses for the groupers until 90 postvaccinated days Thereby, it is the scientific basis for using the recombinant T4 protein antigens as the materials in the production of vaccines against VNN in groupers The structure of the dissertation: The main thesis consists of 105 pages with 16 tables, 32 figures It is divided into five chapters as follows: Introduction: pages, Chapter Literature overview: 34 pages, Chapter Research methodology: 19 pages, Chapter Results and discussion: 47 pages Conclusions and recommendations: pages The thesis includes 71 references, in which there are 15 Vietnamese and 56 English documents CHAPTER LITERATURE OVERVIEW 1.1 Biological characteristics of groupers: Groupers belong to the Serranidae family, Epinephelus genus According to the Institute of Oceanography Nha Trang, Vietnam has about 30 species of groupers (Le Anh Tuan, 2004) [11] Groupers live in warm waters, the temperature suitable for grouper development is from 22-32ºC, the most suitable is 25-30ºC Figure 1.1 Morphology of brownspots grouper (Epinephelus Fish tolerate salinity is from 11 to 41 ‰ coioides) Groupers have mechanical barriers such as oily fluid, skin and gills which protecting fish body against the intrusion of pathogens (Kim Van Van and Le Thanh Hoa, 2009) [12] Groupers have a specific immune system, so when antigens enter the body, they are capable of producing specific antibodies against antigens, protecting them from harmful effects of pathogens 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 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 contains RNA1 (3.1 kb) which encodes a 100 kDa protein that functions as an RNA polymerase The small subspecies contains RNA2 which possesses two high conservation areas, T2 (870 bp) and T4 (420 bp) (Nishizawa et al., 1997) 1.5 Some preventation methods of VNN in groupers Now a day, there is no vaccine to efficiently prevent VNN in groupers Therefore, main preventation methods are to ensure hygiene and to avoid infection sources from fish 1.5.1 Recombinant antigens and vaccine use for fish disease prevention 1.5.2 Recombinant antigens of pathogens A recombinant antigen is an antigen (immune protein) produced by genetic technology In the world, the use of recombinant antigens to produce vaccines that control some serious fish diseases has been studied since the 1980s The application of biotechnology (e.g, the recombinant method) to produce large quantities of antigens has helped to save production costs and increase safety for cultured fish Recombinant DNA technology, which used in antigen production to against viral diseases in fish, has been developing dramatically 1.5.3 The current status of vaccine use for fish disease prevention in the world There are few inactivated vaccines used to prevent viral diseases in fish such as pancreatic necrosis (IPNV), haematopoietic necrolysis (IHNV), hemorrhagic septicemia (VHSV) Spring carp blood virus (SVCV) in the world However, the cost of cell culture in viral diagnosis is relative high and the cell purification is difficult Recently, the use of recombinant DNA technology to produce vaccines from viral proteins using for fish disease preventation is very usefull, highly economical and effective (Christie et al., 1997; Lorenzen et al., 2005) [26] [43] 1.5.4 The current status of vaccine use for fish disease prevention in Viet Nam Since 1996 - 1998, Bui Quang Te et al produced vaccines from Aeromonas hydrophila to prevent diseases for grass carps, with an efficiency of 90 to 100% (Bui Quang Te et al, 2006) [7] In 2003, Vu Dung Tien developed vaccines from the combination of intracellular and extracellular antigens of Aeromonas hydrophila bacteria to prevent 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 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 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) - Orange-spotted groupers (Epinephelus coioides), tiger groupers (Epinephelus fuscoguttatus) humpback groupers (Cromileptes altivelis) at different sizes; - Orange-spotted groupers (Epinephelus coioides), fingerling, juvenile stages (body length 1,5 – cm) - Medium, equipment for cell culture: Leibovit, MEM, HANK's, FCS, antibiotics - Pure chemicals for histopathological test; The chemicals used in the molecular biology are of Sigma, Bio-Basic, Invitrogen, Biological: IPTG, ampicillin, X-gal, agarose, EDTA, SDS, yeast, peptone, Ethidium Bromide, Ethanol, Isopropanol, Sodium Acetate, Acetic Acid, Glycine, Methanol, TBST, BSA, TBS, Tris HCl, EDTA, TAE, NaOH, T4 ligase 2.2 Research contents: - Identification of grouper NNV in Viet Nam; - Investigation of some biological characteristics of grouper NNV; - Creation of the recombinant antigens using as materials to produce vaccines agianst VNN for groupers 2.3 Research methodology 2.3.1 Identification of grouper NNV - Sample collection and processing: 51 grouper samples that doubted of VNN in Quang Ninh (QN1-QN8), Hai Phong (HP1-HP11), Nam Dinh (ND1-ND11), Khanh Hoa (KH1-KH9), Binh Thuan -B12) were collected and refrigerated Disease infected fish samples were then used for collecting brain and eyes - Histopathological method: according to OIE, FAO (2005) - Viral identification by cell culture according to Q.W.Qin et al (2006) - Total RNA extraction of the VNN infective samples by using Qiagen Kit RNeasy Qiagen - 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 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 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 temperature levels: 17, 22, 27 and 32oC The infection dose was TCID50 = 106,8 Testing and evaluating of CPE were performed after 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 Leibovit"z 15 used as controls Fish were raised at 28oC (all day and night) and 28oC (daytime)/ 24oC (at night) Mortality of experimental fish and T4 gene were assessed and identified after 15 days 2.3.3 Expression of antigen-coding genes and evaluation of the antibody producing ability of recombinant antigens - Preparation of pET32a+ vector to perform a T4 gene linked reaction (pET32a+-T4) Using the temperature shock to put pET32a+-T4 vector into E coliBL21 cell - Extraction of plasmids from colonies after transformation - Test PCR and restricted enzyme to determine T4 gene in plasmids - E coliBL21 culture that containing recombinant pET32a+-T4 plasmid - SDS-PAGE electrophoresis - Western blot: to identify T4 recombinant protein that is specific for NNV - Assessing ability to produce antibody of recombinant antigens + Assessing ability to produce neutralizing antibody of recombinant antigen in rabbit: Positive control: rabbit serum without specific antibody was incubated with QN2 (10 TCID50) and adsorbed onto GS1 cells or infected with small groupers (≤ g) by injection (dose of 0,05 ml of poisonous NNV QN2 with titres 103 LD50) Negative controls: rabbit serum was immunized with E.coliBL21-pET32a+-T4 and recombinant T4 protein, diluted with a factor of 10 then adsorbed onto GS1 cells or infected into juvenile groupers (≤ g) by injection Experiment plot: rabbit serum was immunized with E.coliBL21-pET32a+-T4 and recombinant T4 protein, diluted with a factor of 10, incubated with QN2 at 104 TCID50 and adsorbed onto GS1 cells or infected into juvenile groupers (≤ g) by injection (0,5 ml dose of QN2 virus titrated 103 LD50) + Evaluate ability producing the neutralizing antibody of recombinant antigens on grouper Positive control: specific pathogen free fingerling groupers were milled, treated with antibiotics, filtrated and diluted with a factor of 10, incubated with QN2 with 10 TCID50 and infected into GS1 cells Negative controls: specific pathogen-free fingerling groupers were immunized with E coliBL21-pET32a+-T4 and recombinant T4 protein antigen with doses (0.2 mg/ dose) for every 15 days within a period 90days Samples were periodically collected every 15 days, then milled, antibiotic treatmented, filtered, dilutted with a factor of 10 and infected into GS1 cells Experiment plot: specific pathogen free groupers were immunized with E coliBL21pET32a+-T4 and recombinant T4 protein with doses (0.2 mg / dose) for every15 days within a period 90 days Samples were periodically collected, milled, antibiotic treatmented, filtrated, dilutted and incubated with QN2 strain (104 TCID50) and infected into GS1 cells, evaluated CPE for days CHAPTER RESULTS AND DISCUSSION 3.1 Identification of NNV in groupers in Viet Nam 3.1.1 Screening NNV infected fish samples by histopathology The screening results of 51 grouper samples infected NNV by histopathology (Figure 3.1 and Table 3.1) show that there were 26 specimens had vacuole in the eyes and brain tissues The vacuoles in the tested samples are either round or elliptical, with sizes range from 5-10 μm Figure 3.1 Cytopathogenicity in brain and eyes tissues (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 BT2, BT4, BT9, BT12 37,50 27,27 45,45 22,22 33,33 the 26 samples collected The remaining samples had no vacuole, however, we could not rule out the possibility that fish were in the pre-infection Therefore, the molecular biology method was employed to identify antigenencoding genes of NNV 3.1.2 Identification of fish samples infected with NNV by RT-PCR method Bảng 3.2 Identification of NNV by RT-PCR Samples were positive for T4 gene of NNV Ratio (%) (27 samples) QN2, QN4 HP2, HP4, HP6, HP7, HP8, HP10, HP11 ND1, ND3, ND4, ND5, ND7, ND8, ND10, ND11 KH4, KH5, KH9 BT2, BT3, BT4, BT7, BT9, BT10, BT12 Average 25,00 63,63 72,72 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 days NNV infection, granulocyte and rounded cells, B: The cells after days NNV infection, C: Vacuolation in GS1 cells after days NNV infection From day 2, the cells appeared to have a granular, rounded, multi-particle acuolation shape; after days, the cells were died throughout the culture plates and all cells were destroyed at day (Figure 3.3) Figure 3.7 Comparison of the similarity of sequenced genes with T4 gene of Betanodavirus (HM017077.1) The results of T4 gene sequencing led us to conclude that the virus strain identified as Nervous Necrosis Virus belongs to Betanodavirus family 3.2 Investigation of some biological characteristics of NNV 3.2.1 Investigation of pathogenic characteristics of NNV in cells Bảng 3.5 Pathogenic characteristics of NNV in GS1 cells Tissue culture infectious dose of 50% tế bào (TCID50) No Sample ID 1st 2nd 3rd 4th Avarage 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 10-4,3 10-4,9 10-4,8 HP10 10-6,8 10-6 10-3,9 10-6,9 10-5,8 10-4 10-6,8 10-5,7 10-3,9 10-7 10-6,1 10-3,8 10-6,8 10-5,9 10-3,9 ND1 ND3 ND4 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 QN2 HP2 HP4 HP7 HP8 10 11 QN4 HP6 11 No Tissue culture infectious dose of 50% tế bào (TCID50) Sample ID 1st 2nd 3rd 4th Avarage 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 10-4,8 10-3 10-4,9 10-3 10-4,9 10-3 10-4,9 10-2,9 10-4,9 10-3 10-3 10-2,7 10-3,1 10-2,8 10-3 10-2,7 10-3 10-2,6 10-3 10-2,7 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 10-4,7 10-5,9 10-4,8 10-5,8 10-4,8 10-5,9 10-6,8 10-6,9 10-6,8 10-6,7 10-6,8 10-3,9 10-2,5 10-4,8 10-4 10-2,7 10-4,9 10-3,9 10-2,9 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 BT3 BT4 BT7 BT9 BT10 BT12 The experiments show that 6/26 strains are highly virulent with TCID50 dose of 10-6,8 to 106,9 ; 10/26 strains are mildly virulent with a TCID50 dose of 10-4,8-10-5,9 at a dilution of 10-8 and the remaining 10 strains are less virulent with TCID50 doses of 10-2,7-10-4,3 3.2.2 Investigation of pathogenic characteristics of NNV in groupers The study choosed high virulent strains (QN2, QN4, HP7, ND1, KH5 and BT7) and low virulent strains (KH4, BT10) to infect in groupers Fish were checked for days to determine mortality rates and T4 gene presence The results are presented in the table 3.6 Table 3.6 Pathogenic characteristics of NNV in groupers 50% last dead dose of fish (LD50) No Sample ID 1st 2nd 3rd Avarage -6,2 -6,2 -6,1 10 10 10 10-6,2 QN2 10-7,5 10-7,6 10-7,5 10-7,5 QN4 10-7,5 10-7,5 10-7,4 10-7,5 HP7 10-5,4 10-5,6 10-5,5 10-5,5 ND1 10-6,3 10-6,2 10-6,4 10-6,2 KH5 10-6,5 10-6,5 10-6,4 10-6,5 BT7 10-3,7 10-3,6 10-3,6 10-3,6 KH4 10-2,5 10-2,7 10-2,5 10-2,6 BT10 ND ND ND ND Control (Note: ND Not done) Results show that: strains of QN4 and HP7 have a high virulence with LD50 of -7,5 10 , low virulent strains with LD50 from 10-2,6-10-3,6 are KH4 and BT10 3.2.3 Effects of temperature on pathogenicity of NNV in cells 12 GS1 cells were infected with the QN4 strain of NNV with a titre of 10-6,8 TCID50 / ml and cultured for days to determine survival of the cells 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 days, 100% after 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 - - 86,6 83,3 76,6 82,1 3,3 + - 100 100 100 100 6,6 + - 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 (daytime) and 24°C (night time) Time Mortality rate of groupers (%) T4 gene determination (day) 1st 2nd 3rd Average Control Treatment Control 12 15 80,0 100 86,6 100 90,0 100 85,5 100 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 days and 100% after 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 cutting was performed at 37oC for + hours, the product was Figure 3.9 Diagram of pET32a -T4 recombinant vector creation electrophoresed on 1% agarose gel The results of cutting pGEM-T-T4 vector and pET32a+ vector are shown in the figure 3.10 Figure 3.10 pGEM-T-T4 vector (A) and pET32a+ vector (B) were cut by EcoRI enzyme Well 1,2,3,4 (A): cutting products of pGEM-T-T4 plasmid ; Well 1,2,3 (B): cutting products of pET32a+ plasmid ; Well M: Marker 1kb plus 14 The results in the figure 3.10 (A) show that there were two bands in wells 1, 2, 3, and 4, of which one band has 420bp in size (T4 gene size), and another band has a size of approximately 3015bp (similar to pGEM -T Easy vector size) Figure 3.10 (B) shows that in wells 1, 2, 3, only a single band with the size is equivalent to the vector pET32a + which is 5900bp Thus, it is demonstrated that the recombination plasmid pGEM-T-T4 and pET32a+ plasmid were successfully cut to produce compatible sites for inserting T4 gene into the pET32a+ vector The bands of 420bp (containing T4 gene) and 5900bp (containing pET32a+ vector) were cut and purified The cutting products of pGEM-T-T4 plasmid and pET32a+ vector were electrophoresed on agarose gel 1%, the results are shown in the Figure 3.11 Figure 3.11 Purification of cutting products of pGEM-T-T4 plasmid and pET32a+ 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 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 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 Thus, it is possible to confirm that the recombinant pET32a+- T4 vector has been successfully designed Figure 3.14 Electrophoresis of PCR products to test the presence of T4 gene in recombinant vector Well 1-6: PCR products of pET32a+-T4 plasmids, Well M: 1kb Plus ladder; Well DC: PCR product of plasmid pET32a+ 3.3.2 Expression of T4 antigen-encoding gene of NNV 3.3.2.1 Creation of the E coliBL21(DE3) strain carrying antigen-encoding gene of NNV After successfully creating the pET32a+-T4 recombinant vector, four of the six bacterial strains were selected to transform into the E coli BL21 (DE3) strain Transgenic bacteria were cultured on agar LB medium (supplemented Ampicillin with 100μg / ml) at 37°C for 12 to 16 h, Figure 3.15 A colony plate after transforming results shown in the figure 3.15 pET32a+-T4 into E coliBL21 16 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 to 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 Figure 3.18 SDS-PAGE gel polyacrylamide Figure 3.19 SDS-PAGE gel gel 15% of line and polyacrylamide gel 15% of line and Well 1,5: proteins of and lines before Well 1.5: protein of and lines before adding IPTG; Well 2,3,4: protein of line adding IPTG; Well 2,3,4: protein of line after adding IPTG for 2h, 4h, 6h; Well 6,7,8: after adding IPTG for 2h, 4h, 6h; Well protein of line after adding IPTG for 2h, 4h, 6,7,8: protein of line after adding IPTG 6h Well M: Broad-way Multi prestained for 2h, 4h, 6h Well M: Broad-way Multi protein marker prestained protein marker The results in the figure 3.18 show that there was no band of strange proteins on wells and 5, but a band with a size of 17 kDa appeared on Well 2,3,4 and a bold band of approximately 25 kDa, which is similar with the theoritical size of T4 protein on well 6,7,8 Thus, it is possible to concluded that T4 gene has been successfully expressed in the line The results in the figure 3.19 show that there was no band of strange protein in wells and 5, however, a bold band with a size of approximatley 25 kDa, which is similar to the theoritical size of T4 protein antigen, appears in well 2,3,4,6,7,8 The above findings revealved that T4 gene has been successfully expressed in E coliBL21(DE3) bacteria of BL21-pET32a+-T4 lines 2, 3, 4; the recombinant protein had a size of approximately 25 kDa, which is similar to the theoritical size of the T4 protein antigen Protein expression of the T4 gene was then 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 and collected after induction of 3, and 12 h The results (Fig 3.21) show that the samples collected after 3h and 12h give lower a Figure 3.21 T4 recombinant protein was synthesized by the protein content, while time (Well M: standard protein; Well 1, 2, 3: the the samples collected recombinant protein was synthesized after 3, 4, 12h) after 4h give the highest protein content - Determination of culture temperature E coliBL21-pET32a+-T4 strain was cultured at 28oC, 30oC and 37o and sampled after 4h induction The results in the figure 3.22 show that there was no protein band of 25 kDa size observed at culture temperature conditions of 28°C and 30°C, however, at 37°C, the recombinant protein T4 was seen to be Figure 3.22 Effects of temperature on T4 recombinant protein expression (wells 1, 2, 3: protein contents when cultured at expressed 28oC, 30oC and 37oC, Well M: standard protein) 19 - Determination of the concentration of IPTG induction IPTG was added to the culture medium at concentrations from to 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 gene was best expressed mM to mM when the strain was inducted at mM IPTG 3.3.3 Purification of recombinant antigen The results in the figure 3.24 show that all purified strains appeared only one protein band of 25 kDa size, indicating that the T4 protein was successfully purified The study used this T4 protein antigen to test the ability of neutralizing antibodies to produce vaccines for diease Figure 3.24 Electrophoresis of purificated T4 protein on SDSPAGE gel (Well M: standard protein, wells 1-5: T4 antigen in prevention in grouper phase 1, 2, 3, 4, 5) 3.3.4 Evaluation of ability to create antibodies of the recombinant antigen 3.3.4.1 Evaluation of ability to create antibodies of the recombinant antigen in rabbits The ability to create a neutralizing antibody of E coliBL21-pET32a+-T4 antigen and the recombinant T4 protein in rabbits are shown in the table 3.10 and table 3.11 20 Table 3.10 The titer of neutralizing antibody of NNV in vitro Time to collect serum in rabit after immunization (day) Positive control (CPE %) (CPE %) E.coli BL21- Recombinant pET32a+-T4* T4 protein* 98 15 98 25 Negative control Dilutions of rabbit serum were immunizated to neutralize the dose 104 TCID50 E.coli BL21pET32a+-T4* Recombinant T4 protein* ND ND 0 1: ND 98 0 1: 200 1: 50 35 98 0 1: 800 1: 200 45 98 0 1: 800 1: 200 60 98 0 1: 400 1:100 Note: ND (Not done) Table 3.10 shows that in the negative controls, samples did not cause cytopathogenic effects, while in positive controls, normal rabbit serum were not able to neutralize the virulence of QN2 strain, therefore, all cells in culture wells had cytopathogenic effects On 15th day, only the treatment of rabbit serum immunizated E coli BL21pET32a+-T4 produced antibody at a dilution 1:5 In both treatments, antibodies were produced from 25th day after immunization The rabbit serum injected with E.coliBL21pET32a+-T4 had a higher neutralization (at dilution of 1: 200) compared to the serum injected with recombinant protein (at dilution: 1:50) The neutralized antibody titer reached the highest in both treatments from 35th to 45th days, at a dilution of 1: 800 for the serum injected with E coliBL21-pET32a+-T4 and 1:200 for the serum injected with T4 protein On the last day of experiment (60th day) the neutralized antibody titers of two treatments were decreased The serum injected with E.coliBL21-pET32a+-T4 had a neutralized titer at a dilution of 1: 400, while the other, which injected with recombinant T4 protein, had a neutralized antibody titer at a dilution of 1: 100 Results of the determination of neutralized antibody titers of NNV in vivo are shown in the table 3.11 21 Table 3.11 Neutralization antibody titers of NNV in vivo Time to collect Positive control serum in rabit after immunization 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 100 100 0 ND + ND + 15 100 100 0 ND + ND + 25 100 100 0 1: 100 - 1: 50 - 35 100 100 0 1: 100 - 1: 50 - 45 100 100 0 1: 100 - 1: 50 + 60 100 100 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 22 Table 3.12 The titer of creating immune responses of groupers against NNV in vitro Dilutions of extracted fish fluid immunized to neutralize NNV of QN2 strain (104 TCID50) Experiments 30th 45th 60th 75th 90th 15th day day day day day day Positive controls + + + + + + The fish extracted fluid was immunizated with E + coliBL21-pET32a -T4 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 up to 90 days in both treatments This is the scientific basis to produce vaccines against VNN in groupers Up to date, only few vaccines have been used to prevent dieases for high economic value fish speices such as Salmon, Sea bass, Grouper, Seabream, Plaice and most of these vacines are inactive In Japan, Yamashita et al (2009) tested on inactivated vaccines to prevent VNN for 7-dotted groupers (average weight of 25.4 g/ fish) Antibodies were produced after 20 days of vaccination, while those in the control groups did not produce antibodies [69] Pakingking et al (2010) used inactivated vaccines of RGNNV strain to prevent VNN for tiger groupers in commercial culture The results showed that fish immunized with a neutralizing antibody produced antibodies from 15th day (a titre of 1: 800) to 190 th days (a titre of 1: 400) [51] 23 In Vietnam, inactive vaccines with alum glue adjuvants have been effectively used to prevent VNN for groupers in commercial scales with a protection rate of 83% in fish fry and 82% in groupers cultured in cages and ponds (Pham Thi Tam et al., 2015) [6] CONCLUSIONS AND RECOMMENDATION CONCLUSION In this study, we identified 26 strains of NNV in groupers in Vietnam All identified NNV belong to Betanodavirus genus The viruses were capable of causing cytopathogenicity in GS1 cells In which, there were highly virulent strains (TCID50:10-6,8 to 10-6,9), 10 mildly virulent strains (TCID50: 10-4,8 to 10-4,9) and 10 less virulent strains (TCID50: 10-2,7 to 10-4,3) The viruses had an ability to cause other diseases in groupers, the study identified two highly virulent strains (LD50: 107,5 ), mildly virulent strains (LD50: 10-5,5-10-6,5) and less virulent strains (LD50:10-2,6-103,6 ) The viruses were best adapted and developed when cultured at 22oC.Temperature affects the pathogenicity of the viruses in groupers The mortality rates of experimental fish were 82.1 - 85.5% after days and 100% after days in both experimental conditions, constant at 28oC (all day) and variation at 28oC (daytime) and 24oC (night) The project sucessfully created the recombinant T4 antigen and expressed it into E coliBL21(DE3) cells with a size of 25 kDa The recombinant T4 protein has an ability to produce immune responses for groupers with a neutralizing antibody titer of 1: 128 between 30th and 45th days; the produced antibody lasts until 90th day of the experiment This is the scientific basis for application of the recombinant T4 protein antigen in the production of vaccines against dieases in groupers RECOMMENDATION Further research on optimization conditions is necessary to obtain the highest T4 protein content for producing a high amount of antigens using as materials in the production of vacines in the future Evaluation of immune response ability of cultured fish at fingerling and juvenile stages to determine a suitable protection time of recombinant antigens Based on findings of this research, we recommend to conduct experiments on producing a new generation of recombinant vaccines to prevent VNN in groupers in Vietnam 24 LIST OF SCIENTIFIC WORKS PUBLISHED RELATION WITH DOCTORAL THESIS Pham Thi Tam, Pham Cong Hoat, Nguyen Thi Thu Hien, Vu Tien Lam, Nguyen Thi Vui, Nguyen Thi Thanh (2012), “Study on the pathogenicity and immune response of Nervous Necrosis Virus (NNV) in grouper culture in Vietnam”, Veterinary sciences and techniques, No 6, page 81-88 Nguyen Thi Thanh, Pham Thi Tam, Pham Cong Hoat, Le Van Nam, Tran The Muu, Nguyen Quang Linh (2013), “Evaluation of abitity on inducing immune response of nervous necrosis virus antigen”, Veterinary sciences and techniques, No 5, page 30- 36 Nguyen Thi Thanh, Pham Thi Tam, Man Hong Phuoc, Pham Cong Hoat, Le Van Nam (2014), “Expression of antigen encoding gene of Nervous necrosis virus(NNV) in grouper”, Veterinary sciences and techniques, No 5, page 26-32 ... tables, 32 figures It is divided into five chapters as follows: Introduction: pages, Chapter Literature overview: 34 pages, Chapter Research methodology: 19 pages, Chapter Results and discussion:... in Vietnam It identified 26 viral strains and their biological characteristics - This is also the first research in Vietnam to successfully create the recombinant antigen, T4 protein, which can... 2015) [6] CHAPTER 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