Tom Tat Tieng Anh Hoan Chinh.pdf

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIET NAM ACADEMY OF AGRICULTURAL SCIENCES MAN HONG PHUOC RESEARCH PRODUTION AN INACTIVATED VACCINE TO PREVENT NERVOUS N[.]

AND TRAINING AGRICULTURE AND RURAL

DEVELOPMENT

VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

-

MAN HONG PHUOC

RESEARCH PRODUTION AN INACTIVATED VACCINE TO PREVENT NERVOUS NECROSIS DISEASE IN GROUPER

VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

Scientific instructors:

1 Assoc Prof Ph.D Pham Thi Tam

2 Assoc Prof Ph.D Dong Van Quyen

Review 1: Assoc Prof Ph.D Vo Thi Bich Thuy

Review 2: Assoc Prof Ph.D Kim Van Van

Review 3: Assoc Prof Ph.D Nguyen Quang Huy

The thesis was defended at the Institute's Thesis Evaluation Council at the Vietnam Academy of Agricultural Sciences

Time: , date:

The thesis can be found at the library:

1 National Library of Vietnam

2 Library of Vietnam Academy of Agricultural Sciences

INTRODUCTION

1 RATIONALE OF THE STUDY

The aquaculture industry is considered one of the key economic sectors of Vietnam and has continuously grown in recent years in terms of both area and output Total seafood production in 2022 is estimated at 9,026.3 thousand tons, up 2.7% compared to 2021, of which aquaculture production is estimated at 5,163.7 thousand tons, up 6.3% compared to

2021 However, the aquaculture industry in the world as well as in Vietnam always faces difficulties and challenges, especially in recent years, viral diseases in aquatic animals such as zoonotic diseases grass carp haemorrhage, spring virus disease in carp, grouper Iridovirus disease, infectious pancreatic necrosis, infectious spleen and kidney necrosis, infectious haematopoietic necrosis, viral hematoma causing serious economic losses to aquaculture

Viral nervous necrosis (VNN) is detected in most countries around the world, affecting more than 120 marine fish species, many of which are

of high economic value The disease has been determined to be caused by

a nervous necrosis virus (NNV) Fish suffering from nervous necrosis disease appear with typical symptoms: swimming without orientation, dark body, anorexia, 80-100% of infected fish may die after 3-5 days

Diseases in aquaculture tend to increase over the years, with a wide range of infections, high incidence rates, diverse types, and long onset times Aquaculture is an urgent problem Disease control by microbial products, immunostimulants, and vaccines is increasingly being applied in ecological farming models In particular, using vaccines is an effective and safe preventive measure to prevent diseases caused by viruses and bacteria

Currently, most commercial aquatic vaccines are still inactivated vaccines because of their safety The virus causes severe neurological necrosis disease in the larval, fry, and fingerling stages with a relative percent survival up to 100%, therefore, the vaccine against nervous necrosis disease is often used for fish by immersion vaccination

Stemming from the above theoretical basis and practical needs, the topic: "Research production of inactivated vaccines against nervous

necrosis disease for grouper (Epinephelus spp.)" was conducted to create

inactivated vaccine against neurodegenerative disease in grouper

Producing inactivated vaccine against nervous necrosis disease for grouper

Develop a process to check vaccine quality

3 SCIENTIFIC AND PRACTICAL SIGNIFICANCE OF THE THESIS

3.1 Significance scientific

The thesis has produced an inactivated vaccine to prevent nervous necrosis disease in grouper with the following steps: isolation, selection of seed strains as raw materials for vaccine production, evaluation of the original strains, and virus culture on cells, inactivate the virus, determine the conditions for the production of semi-finished vaccines, and evaluate the quality of the semi-finished vaccines The research results are scientific data, providing more references for the production of inactivated vaccines against diseases for marine fish with high economic value

3.2 Significance practical

The thesis has successfully researched the inactivated acacia alum vaccine with safety and efficacy criteria in the prevention of nervous necrosis in grouper

4 Object and scope of research

4.1 Research subjects:

Viral strains causing nerve necrosis were isolated from some species

of grouper farmed in some provinces in the North

4.2 Research scope:

Virus strains that cause neurological necrosis disease were isolated from marine fish cultured in the waters around Cat Ba Island, Lan Ha Bay, Cat Hai (Hai Phong), Van Don, Quang Yen (Quang Ninh), and marine areas Dong Chau (Thai Binh), Hai Thinh, Nghia Hung (Nam Dinh)

5 CONTRIBUTIONS OF THE NEW THESIS

The thesis has comprehensively studied the characteristics of the original seed strain for the production of vaccines against nervous necrosis disease in grouper, including purity, protective immunogenicity, and stability Genetics and yield after culture on GS01 cells

The thesis has made a colloidal alum inactivated vaccine by TB05 virus isolated in Thai Binh province, alum inactivated vaccine is used for fish by immersion vaccination, 100% safe rate, relative percent survival 75.8% after 3 months

6 LAYOUT OF THE THESIS

The thesis consists of: Introduction 5 pages, Overview 27 pages, Objects and research methods 16 pages, Research results 53 pages, Conclusion and recommendations 1 page, 250 references, 22 tables, 18 pictures

Chapter 1 DOCUMENTARY OVERVIEW 1.1 Several biological characteristics of grouper

Groupers belong to the family Serranidae, subfamily Epinephelinae, genus Epinephelus, English name: groupers There are all 87 species listed

in the genus Epinephelus (E.), 63 species of Epinephelus have been

discovered in the Indian and Pacific regions alone, grouper is a dwelling fish in coral reefs and rocks Hard, warm waters, distributed mainly in tropical and subtropical seas and rarely found in temperate seas The suitable temperature for grouper species to grow is 25-30 degree celsius, salinity from 11-41‰ Almost all grouper species are hermaphrodites The gonads were initially undifferentiated, then they were differentiated into ovaries in all individuals After maturing into females, they undergo a sex change into males, transforming the ovaries into testes Natural sex change in grouper species occurs between 3 and 11 months of age, depending on the species Grouper tends to lay eggs in early spring and summer Although some species, such as grouper, breed year-round, most species of grouper breed between January and May

bottom-1.2 Epidemiology of nervous necrosis disease in grouper in the world and Vietnam

Viral nervous necrosis has been officially reported worldwide, with the exception of South America VNN appeared in Asian countries such as India, Indonesia, China, Japan, Korea, Malaysia, Philippines, Thailand, Vietnam; Oceania such as Australia, Tahiti; Mediterranean region such as France, Greece, Italy, Malta, Portugal, Spain, Tunisia or other countries such as, United Kingdom, Norway, Caribbean Islands and North America

In Asia, mass mortality has been recorded in the rearing stages of

grouper in Singapore, red-spotted grouper (E akaara) and striped bass (Pseudocaranx dentex) in Japan, and flathead headfish (Mugil cephalus) in China, as well as red snapper (Sciaenops ocellatus) in Israel In North America, VNN has been detected in white seabass (Atractoscion

nobilisthe) and Atlantic cod In addition, nervous necrosis disease infects

not only marine fish but also many freshwater aquatic animals

In Vietnam, some species of grouper infected with nervous necrosis

disease have been detected, including: E coioides, E fuscogutatus, E tauvina, E lanceolatus, E malabaricus in Khanh Hoa, E coioides in Cat Ba

(Hai Phong) and Cua Hoi (Nghe An) Nervous necrosi disease in grouper in Vietnam was first detected in 2002 in cages at Van Don, Quang Ninh

1.3 An overview of the virus that causes nervous necrosis

The causative agent of neuroleptic disease in fish was identified as

Betanodavirus, Virus without envelope, spherical, 25-30 nm in diameter,

with tetrahedral symmetry, protein coat consisting of 180 proteins with

mass 42 kDa The genome of Betanodavirus has a single-stranded,

positive-stranded RNA structure consisting of two subunits: The large subunit of the genome contains RNA1 (3.1 kb) and the small subunit of the genome contains RNA2 (1.4 kb), on which RNA2 contains a reading frame open (ORF) for an envelope protein that also contains two highly

conserved regions, T2 (870 bp) and T4 (420 bp) The Betanodavirus genus

is classified into 4 main genotypes, including: striped flounder nerve necrosis virus (SJNNV), tiger puffer nerve necrosis virus (TPNNV), grouper nerve necrosis virus red spot (RGNNV) and halibut neural necrosis virus (BFNNV)

The virus enters host cells by means of horizontal and vertical transmission and produces a full-fledged infection by entering sensitive cells of the central nervous system including the brain and spinal cord life and retina leads to clinical manifestations of fish when infected such as: swimming without orientation, dark body, bulging eyes, curved body

1.4 Overview aquatic vaccines

Vaccines in aquaculture are considered an effective preventive measure against many diseases and have recently gained popularity According to the causative agent, aquatic vaccines can be classified into bacterial vaccines, viral vaccines and parasitic vaccines, they can also be classified according to the composition including vaccines monovalent polyvalent or mixed vaccines, or classified by nature such as live attenuated vaccines, inactivated or subunit vaccines, acid vaccines nucleic, recombinant vaccines It should be noted that each vaccine has advantages and disadvantages, and different vaccines need to be developed and used for different pathogens and animals

The method of using vaccines in aquaculture is usually administered

by injection, immersion vaccine and oral vaccine Each type also has advantages and disadvantages and is suitable for different audiences

Chapter 2 MATERIALS, CONTENTS AND METHODS STUDY 2.1 Research Materials

2.1.1 Fish to disease

Grouper samples were collected in coastal aquaculture areas around Cat Ba Island, Lan Ha Bay, Cat Hai (Hai Phong), Van Don, Quang Yen (Quang Ninh), Dong Chau (Thailand) waters Binh), the sea area of Hai Thinh, Nghia Hung (Nam Dinh)

2.1.2 Experimental fish

The orange-spotted grouper (Epinephelus coioides) 2-2.5 cm long, the tiger grouper (Epinephelus fuscoguttatus) 2-2.5 cm long and the mouse grouper (Cromileptes altivelis) 5.5-6 cm long were provided by the Centre

Country Northern seafood varieties, Xuan Dan, Cat Ba, Hai Phong

2.2.2 Producing inactivated vaccine against nervous necrosis disease for grouper

2.2.3 Develop a process to check vaccine quality

2.3 Location and time of study

2.3.1 Location: The study was carried out at the Microbiology laboratory - Faculty of Biotechnology, Hanoi Open University and the Department of Molecular Microbiology - Institute of Biotechnology, Vietnam Academy of Science and Technology

2.3.2 Research period: From April 2016 to April 2020

2.4 Research methods

2.4.1 Sample collection and processing methods

2.4.2 Virus isolation technique on GS01 susceptible cells

2.4.3 Total RNA extraction technique

2.4.4 Reverse Transcriptase PCR (RT-PCR)

2.4.5 PCR product purification method

2.4.6 Agarose gel electrophoresis

2.4.7 DNA sequencing method

2.4.8 Method to determine TCID50, LD50 of NNV

2.4.9 Determining the conditions for virus propagation

2.4.10 Virus inactivation method

2.4.11 Method of making alum colloidal vaccine

2.4.12 Cell neutralization method

2.4.13 Immunization method

2.4.14 Indirect ELISA reaction

2.4.15 Powerful challenge method

2.4.16 Vaccine safety assessment

2.4.17 Evaluation of vaccine efficacy under experimental conditions 2.4.18 Data processing methods

Chapter 3 RESULTS AND DISCUSSION 3.1 Results of isolation and selection of original strains for the production of vaccines against nervous necrosis disease for grouper

3.1.1 Isolation of virus causing nervous necrosis disease from grouper suspected of infection in 4 Northern provinces, Vietnam

In total 60 samples of grouper with symptoms of neuroleptic disease were collected from waters around Cat Ba island, Lan Ha bay area, Cat Hai (Hai Phong), Van Don sea area, Quang Yen province Quang Ninh), Dong Chau sea (Thai Binh), Hai Thinh and Nghia Hung (Nam Dinh)

Figure 3.1 Samples of grouper suspected of being infected with

nervous necrosis disease

A: healthy grouper; B and C: sample of grouper suspected of having VNN

Table 3.1 Results of screening for T4 gene in fish samples suspected of

being infected with NNV Location Number of samples Number of samples

positive for T4 gene

Positive rate (%)

HP 15 6 40,00

Figure 3.2 Electrophoresis images of RT-PCR products of some

samples suspected to be infected with NNV

Wells 1-5: samples suspected of NNV infection; M: DNA ladder

standard 1kb Screening results by molecular biology from 60 samples of grouper suspected of being infected with nervous necrosis disease, 32 samples found that the gene fragment was 420 bp in size, corresponding to the size

of the T4 gene fragment of the virus causing the nervous necrosis disease T4 positive samples were used to isolate the virus on GS01 susceptible cells

Viruses multiply in cells and are assessed by cytolytic effects (CPE)

Figure 3.3 Image GS01 cell

A: GS01 cells have not been infected with the NNV sample

B: GS01 cells after 120 hours of infection with NNV

Table 3.2 Results of culture of patient samples on GS01 cells Nume

rical

order

Sample symbol

Cytopathogenic effect (CPE) over time (hours)

Notes: ++++: CPE > 98%; +++: CPE > 85%; ++: CPE > 50%; +: CPE >

20%; +: suspected CPE > 10%; -: no CPE The results of virus isolation on GS01 cells showed that, in GS01 cell culture dishes, the patient samples started to cause cell damage after 72 hours of infection, showing increased cell damage in the next days and peak from 168 to 192 hours after infection

3.1.2 Determination of virulence (TCID 50 and LD 50 )

Viral virulence was determined by the tissue culture infective dose (TCID50) and/or the dose that was lethal dose to 50% of the laboratory animals (lethal dose (LD50)

Table 3.3 Determination results TCID 50 on GS01 cells Nume

rical

order

Sampl

e symbol

Cytopathogenic effect (CPE) (%) according to virus

LD50/mL, two highly virulent samples, TB05 and HP02, were selected for species identification by molecular biology method

The results of species identification of two samples TB05 and HP02 through T4 gene sequencing with RNA2 sequences of nerve necrosis virus strains were published on Genebank This result allows to conclude that TB05 and HP02 samples are nervous necrosis virus

Table 3.5 Compare the similarity of the T4 gene sequences of HP02 and TB05 samples with those of GenBank

Code GenBank Strain name and

sequencing gene

Percentage

of sequences compared to GenBank (%)

MG874758.1

Epinephelus coioides nervous necrosis virus isolate HN1 segment ARN2, complete sequence

AF283554.1

Yellow grouper nervous necrosis virus major coat protein gene, partial cds