Phân lập, định danh virus viêm gan vịt ở một số tỉnh đồng bằng sông cửu long và sản xuất kháng thể phòng bệnh tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Microbiology Code: 62 42 01 07 PHẠM CÔNG UẨN ISOLATION, IDENTIFICATION OF DUCK HEPATITIS VIRUS IN SOME PROVINCES IN THE MEKONG DELTA AND PRODUCTION OF ANTIBODIES TO PREVENT DISEASE Can Tho, 2019 The thesis was completed at Can Tho University Scientific supervisors: Assoc Prof Dr Ho Thi Viet Thu Reviewer Assoc Prof Dr ……… Reviewer 2: Assoc Prof Dr Reviewer 3: Assoc Prof ……… The thesis is defended in front of the University Examination Council in Can Tho University Time:…… March ……, ………… COUNCIL CHAIRMAN Assoc Prof Dr………………… Further information of the thesis could be found at: Learning Resource Center of Can Tho University National library of Vietnam PUBLIC WORKS Pham Cong Uan and Ho Thi Viet Thu, 2014 Isolation and identification of duck hepatitis virus type in Hau Giang Province Can Tho University Scientific Journal Special subject of Agriculture, 2:116-121 Pham Cong Uan and Ho Thi Viet Thu, 2016 An examination on the virulence and the pathogenicity of a duck hepatitis A virus strain genotype isolated in Hau Giang province Can Tho University Scientific Journal Special subject of Agriculture, 42:7-10 Pham Cong Uan and Ho Thi Viet Thu, 2018 Examination on pathogenicty of duck hepatitis virus isolated from ducks in Hau Giang Province Can Tho University Scientific Journal Special subject of Agriculture, 54 (1B): 1-6 BEGINING PART CHAPTER INTRODUCTION 1.1 The urgency of the study Duck viral hepatitis is a highly fatal disease with high mortality, it rapidly spreads in ducking flocks, especially in the Mekong Delta, but until now there has not had any research about this disease implemented in the Mekong Delta So, the disease remains the major threat to the health of ducks as well as affecting the economic result in the region So that the determination of major pathogenic viral strains, and using them as antigens in vaccine and antibody production is necessary requirements in for the strategy of specific prevention and treatment in order to protect duck health, to respond to the urgent request the study: "Isolation and identification of duck hepatitis virus in some provinces in the Mekong Delta and production of antibodies to prevent disease" was carried out 1.2 Objectives of the study The goals of the study are: To isolate and determinate viruses causing hepatitis outbreak in ducks from the field To study the genetic relationship about the origins of the hepatitis viruses isolated from diseased ducks To produce IgY antibodies from chicken egg yolk and examine its efficacy in prevention and treatment of duck viral hepatitis 1.3 Research content Contents 1: Isolating and identifying the viruses causing hepatitis for ducks in provinces in the Mekong Delta Content 2: Study on the genetic relationship of the hepatitis duck virus strains isolated in provinces in the Mekong Delta Contents 3: Surveying of pathogenicity of represented DHAV-3 virus strain, symbol: DHAV-3-HG2 Content 4: Production of duck hepatitis virus antibodies by IgY antibody production technology from chicken yolks and its application in prevention and treatment 1.4 Time and place of study The study was carried out from 2012 to 2016 Suspicious viral hepatitis ducks were collected from Can Tho city and Vinh Long, Tra Vinh, Kien Giang, Hau Giang province Isolation and identification were done in virology laboratories and molecular biology laboratories of Can Tho university Nucleotide sequence was carried by Macrogen cooperation (South Korea) 1.5 New contributions of the thesis DHAV-3 is determinated the main cause of duck viral hepatitis in the Mekong Delta DHAV-3 was quite nucleotide homogeneous and separated into group compared to other DHAV-3 strains in the other countries and in Asia Successful production of purified IgY antibodies from strains DHAV-3 isolated in the Mekong Delta and it showed effective in treatment and prevention 1.6 The composition of the thesis The thesis is 174 pages and consists of chapters: introduction, materials and methods, results and discussions, conclusions and suggestion, references and appendices There are 31 tables, 38 figures and 119 references documents CONTENT CHAPTER 2: LITERATURE OVERVIEW Duck hepatitis is caused by any of three different viruses, namely duck hepatitis type I, type II and type III Currently, genotypes of duck hepatitis virus type I (DHAV) were identified They are belong to Picornaviridae family, Avihepatovirus genus There are duck virus hepatitis type I genotype (DHAV-1), duck viral hepatitis type I genotype (DHAV-2) and duck hepatitis virus type I genotype (DHAV-3) Duck hepatitis virus type II is classified as Duck Astrovirus type (DAstV-1), viral hepatitis type III is Duck Astrovirus type (DAstV-2) belong to Astroviridae family DAstV-1 is mainly reported in the UK, the DAstV-2 is only reported in the United States (OIE, 2010) Mekong Delta is the most important duck production, duck viral hepatitis occur frequently and cause big loss for duck raiser but there is not research on this disease and vaccine and antibody production For proper prevention and treatment, the identification of the currently circulating strain of the virus is common and is used as an antigen for the hens to produce IgY antibody from egg yolk is necessary to develop optimal measures CHAPTER 3: STUDY METHODS 3.1 Method for isolation, identification and genetic analyzing of virus 3.1.1 Method of isolation of hepatitis virus on duck embryos Ninety two specimens fluid (HDBP) from 92 duck diseased flocks were collected in the Mekong Delta were injected into the allantoic cavity of the 12-day-old duck embryo, the specimens fluid of each duck is injected into one embryo; then follow up: dead time of embryo; gross lesions on the embryo 3.1.2 Methods of inoculating hepatitis virus on duck embryo fibroblast Allantoic fluid and embryonic liver was homogenated in a suspension and continued to be inoculation into duck embryo fibroblasts Then, inoculated embryo fibrioblasts were collected for duck hepatitis virus identification and quantification by RT-PCR 3.2 Genetic analyzation of duck hepatitis virus type I genotype 3.2.1 Sequencing of nucleotides Ten RT-PCR products, denoted DHAV-3-CT2, DHAV-3-CT2, DHAV-3-CT6, DHAV-3-CT6, DHAVDHAV-3-VL8, DHAV-3-VL8 in 63 strain isolates were used for nucleotide sequencing and for genetic analysis 3.2.2 Genetic studies of isolates of DHAV-3 Genetic analyzation of 10 representative strains was performed by comparing nucleotide analogues, amino acids with 31 strains in the World Gen Bank, inside strains were isolated from Vietnam with the GenBank number KU860090.1 , KU860089.1, JF914944.1; strains were isolated from Korea and 23 strains were isolated from China Analysis was performed using BioEdit software and genealogy relationships were established using MEGA 5.1 software 3.3 Virulent examination of strain of DHAV-3-HG2 on embryos and ducklings 3.3.1 Quantification of tissue culture infective dose 50% (TCID50) Experiment was conducted on 96 wells, virus fluid was diluted from 100 to 10-10, each diluted to wells in the same column, each well 100 l minimum essential medium, starting from concentration the highest dilution and control wells, each well 100 l minimum essential medium Calculate the TCID50 by the method of Reed and Muench (1938) Tracking: TCID 50 /0.1ml 3.3.2 Quantification of lethal dose 50% on duck embryos and pathological on the embryos Experimental design: Uses 50 ducks with 12-day-old embryos divided into 10 lots, each containing duck eggs; lots were injected virus fluid from 10-4 to 10-12 and control were injected physiological aqueous solution Tracking: Embryo mortality; ELD50 ; Average embryo death time by concentration; Frequency of appearance of lesions on the embryo 3.3.3 Quantification of lethal dose 50% on ducks and pathological characteristics of virus Experimental design: Complete randomized design with treatments (NT) with replications; There were treatments corresponding to the dilution of 10-1 to 10-5 and control treatment only used physiological aqueous solution, each using ducks The follow-up period was 14 days Tracking: LD50; number of dead ducks; frequency of appearance of symptoms; frequency of appearance of lesions; microscopic lesions on ducks 3.4 Production of anti-DHAV-3 antibody by IgY antibody technology in chickens and use in prevention and treatment 3.4.1 Production of anti-DHAV-3 antibody 3.4.1.1 Create immunity for hens Experimental setting is as Table 3.1, the interval between injections is weeks Table 3.1 Experimental design of hens immunity Experiment Treatment Number of chickens Viral dose (ELD 50/ml) Dose (ml) Route Number of injections 104/1 104 1 10 4/2 104 104/3 104 106/1 106 106/2 106 106/3 106 108/1 108 108/2 108 10 8/3 108 Control PBS Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle 3 3 3.4.1.2 IgY antibody extraction from egg yolk IgY antibody from egg yolk is extracted by method of salt deposition (NH4)2SO4 saturation 40%, then dialysis to remove the salt Post-dialysis solutions were analyzed for protein concentration by Bradford method and quantification of antibody titres IgY was determined by virus neutralization 3.4.1.3 Examination of IgY antibody titres in hen and egg yolk Antibody titres IgY was tested by viral neutralization Follow-up: The antigenic dose generated is best immunized; The number of times the antigen injection causes a prolonged and stable immune response; The correlation between antibody titres IgY in hen serum and egg yolk 3.4.1.4 Quantification safe dose of IgY antibody from egg yolk on duck embryo fibrioblast Experimental design: The experiment was conducted on 96 wells with 10 concentrations of diluted IgY diluted in (1/2, 1/4, 1/8, 1/16, 1/32, 1/64 , 1/128, 1/256, 1/512, 1/1024), the initial IgY concentration was 13.5 mg/ml Each concentration was repeated times Negative control is a duck embryo fibrioblast that does not provide IgY injections but only for the minimum essential medium Follow up: Morphology of cell layer (with or without CPE and CPE levels); OD value after MMT staining (showed the proportion of living cells) 3.4.1.5 Investigation of DHAV-3 resistance activity of IgY antibody on duck embryo fibrioblast at 24h before and after infection Experimental design: The experiment was performed on 96 well wells with four concentrations of diluted IgY antibody for 10 yr from 100 to 10-3 and injected into DEF medium at 24h before and after inoculation DEF with DHAV-3 fluid of dose 500TCID50/0.1ml/well The initial concentration of IgY antibody yolk is x = 1.68 mg / ml, at subsequent concentrations, diluted IgY antibody concentration is 10 Each concentration was repeated 20 times Nagative control is a duck embryo fibrioblast only for the minimum essential medium Positive control is a duck embryo fibrioblast that does not given IgY antibody but given virus fluid at 500TCID50/0.1ml /well Follow up: Morphology of cell layer (with or without CPE and CPE levels); OD value (showed he proportion of living cells) 3.4.1.6 Quanitification protection dose 50% for the 12-day-old duck embryo of yolk IgY antibody The experiment was carried out on 12 day-old duck embryo and disign as Table 3.2 Embryo protection dose 50% (EPD50) ; Three doses of KT IgY is 10EPD50, 30EPD50, 50EPD50 were selected for the prevention and treatment of DHAV-3 virus Table 3.2 Experimental design quanitification of protection dose 50% duck embryos of IgY antibody Antibody dilution Viral dose (LD 50 ) Number of duck eggs have embryos Injection dose (antibody + virus) (ml) Route (10 ) 10 0.1 + 0.1 Allantoic cavity 1/2 (10 -0.3 ) 10 0.1 + 0.1 1/4 (10 -0.6 ) 10 0.1 + 0.1 1/8 (10 -0.9 ) 10 0.1 + 0.1 1/16 (10 -1,2 10 0.1 + 0.1 1/32 (10 -1.5 10 0.1 + 0.1 1/64 (10 -1.8 10 0.1 + 0.1 10 0.1 + 0.1 10 0.1 + 0.1 10 0.1 + 0.1 10 0.1 + 0.1 Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity Allantoic cavity ) ) ) 1/128 (10 -2.1 1/256 (10 -2.4 1/512 (10 -2.7 ) ) ) 1/1024 (10 -3.0 ) 3.4.2 IgY antibody application in prevention and treatment 3.4.2.1 IgY antibody application in prevention A stydy on the efficacy of IgY antibody in prevention of DHAV- virus on ducklings was complete randomized design with dose and repetitions, each treatment used duck Experiment is arranged as Table 3.3 At 24 hours after duckling was supplied IgY by oral and intramuscular injection, the ducks were infected by DHAV-3 virus (DHAV-3-HG2 strain) with dose virus 103.3LD50 Monitoring indicators: Ducks live in treatments after infected by viral hepatitis Table 3.3 Prophylactic experiment from DHAV-3-HG2 with yolk IgY antibody Repeatimes Number of ducks in treatment 10 EPD 50 Dose of virus challenge (0.5ml/unit) 10 3.3 LD 50 30 EPD 50 10 3.3 LD 50 50 EPD 50 KTV 0.5 ml 10 3.3 LD 50 3 5 10 EPD 50 10 3.3 LD 50 30 EPD 50 10 3.3 LD 50 50 EPD 50 10 3.3 LD 50 KTV 0.5 ml 10 3.3 LD 50 5 Treat Road level Dose of antiboy (0.5ml/unit) Oral Oral Negative control Pasitive control Oral Oral Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle 0.5 ml of IgY(-) 0.5 ml of IgY(-) 10 3.3 LD 50 0.5 ml of PBS 10 3.3 LD 50 3.4.2.2 IgY antibody application in treatment A stydy on the efficacy of IgY antibody in treatment of DHAV- virus on ducklings was complete randomized design with dose and repetitions, each treatment used duck Experiment is arranged as Table 3.4 Table 3.4 DHAV-3 therapeutic experiment by KT IgY yolk and KTV antibody Treat The time of KT After 12 hours After 12 hours After 12 hours After 12 hours After 24 hours After 24 hours After 24 hours After 24 hours Pasitive control After 24 hours Negative control After 24 hours Route Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Pectoral muscle Antibody dose (0.5ml ) Viral dose (0.5ml/unit) Times Repeat Number of ducklings 10 EPD 50 103.3 LD 50 30 EPD 50 103.3 LD 50 50 EPD 50 103.3 LD 50 KTV ml 103.3 LD 50 10 EPD 50 103.3 LD 50 30 EPD 50 103.3 LD 50 50 EPD 50 103.3 LD 50 KTV ml 103.3 LD 50 0.5ml IgY(-) 103.3 LD 50 5 0.5ml IgY(-) 0.5ml PBS 3.5 Data processing methods To access world gene bank by the BLAST program Comparison of nucleotide and amino acid by BioEdit software Genealogical scheme set by the MEGA software 5.1 Statistical processing software by Minitab 16.0 Chapter RESULTS AND DISCUSSION 4.1 Results of isolation and identification of hepatitis virus in some provinces of the Mekong Delta 4.1.1 Results of isolation of duck hepatitis virus on duck embryos Table 4.1 showed that, out of 92 samples of virus fluid from 92 duck flocks disease, 78 samples killed the embryos at an average of 84.8% Time to kill embryos from 48-72 hours Table 4.1 Results of isolation of the hepatitis virus on duck embryos Place Number of embryos monitored Embryonic Death Time (hours) Injection dose (ml) Kien Giang Hau Giang Can Tho Vinh Long Tra Vinh Total 18-24 Embryos Ratio dead (%) 24-48 Embryos dead Ratio (%) 48-72 Embryos Ratio dead (%) 72-96 Embr Ratio yos (%) dead Total embryos dead Ratio (%) 22 0.2 0 22.7 11 50.0 13.6 19 86.4 25 0.2 0 28.0 14 56.0 4,0 22 88.0 15 0.2 0 20.0 60.0 13.3 14 93.3 12 0.2 0 25.0 50.0 8.3 10 83.3 18 0.2 0 22.2 44.4 5.6 13 72.2 0 22 23.9 48 52.2 8.7 78 84.8 92 4.1.2 Results of transmission of hepatitis virus on duck embryo fibroblast All 78 samples cause various types of cellular invasions, such as the gradual contraction of the cells, then clustering or joining together to create intercellular cells and eventually necrotic cells forming melancholy 4.1.3 Results of identification of hepatitis virus by molecular biology technique The RT-PCR produce amplifies the 286 bp gene from the nucleotide position of 3,527 to the nucleotide 3,777 of the P2 gene complex in the DHAV-3 genome Fig 4.1 Spectrometry of RT-PCR on agarose gel 1.5% Note: M: Marker size 100bp Wells 1, 2, 3, (KG1, KG5, KG8, KG12) Wells 5, 6, (HG2, HG4, HG5) Wells 8, 9, 10 (CT2, CT6, CT13) Wells 11, 12, 13 (VL1, VL3, VL8) Wells 14, 15, 16 (TV5, TV5, TV8) Table 4.2 showed that, in 78 fluid cell cultures, 56 specimens were positive for primer DHAV-3F, DHAV3R, 71.8% (56/78) of which Kien Giang (84.2%), Hau Giang (81.8%), Tra Vinh (61.5%), Vinh Long (60.0%) and Can Tho (57.1%) This study showed that DHAV-3 is prevalent in duck flocks in provinces in the Mekong Delta DHAV-1, DHAV-2 and the type II hepatitis virus have not been detected Table 4.2 Percentage of positive dengue virus types Place DHAV-1 DHAV-2 (n) (+) %) (n) (+) Kien Giang 19 0 19 Hau Giang 22 0 22 Can Tho 14 0 Vinh Long 10 Tra Vinh 13 Total 78 DHAV-3 (%) DAsTV-1 (n) (+) (%) (n) (+) (%) 19 16 84,2 19 0 0 22 18 81,8 22 0 14 0 14 57,1 14 0 10 0 10 60,0 10 0 0 13 0 13 61,5 13 0 0 78 0 78 56 71,8 78 0 4.2 Results of molecular genetic survey of DHAV-3 strains isolated 4.2.1 Results of nucleotide sequencing of RT-PCR products Sequencing of 10 RT-PCR products out of a total of 56 products resulted in a 240 nucleotide fragment 4.2.2 Comparison of nucleotide and amino acid sequences between 10 strains isolated from other strains in the World Bank DHA-3-CT2, DHAV-3-CT2, DHAV-3-H3, DHAV-3-HG2, DHAV-3 DHAV-3-TV5, DHAV-3-VL8 have nucleotide analogues (98-100%) and amino acids (97-100%); DHAV-3-VL3 had nucleotide analogues (93-94%) and amino acids (89-92%) Fig 4.2 showed that 10 strains isolated in the Mekong Delta formed a separate group and closely related their origin with DHAV-NT isolates in Ninh Thuan province of Vietnam and strains from China DHAV -12-01, DHAV-BN, DHAV-Du-090905 The DHAV-NT, DHAV-DN2 and DHAV-NC were isolated in different parts of Vietnam outside the Mekong Delta in different branches While, in the 23 strains of China and strains of Korea are also classified into different branches of the family tree Fig 4.2 The correlation coefficient between DHAV-3 strains varies with different DHAV-3 strains in the World GenBank Note: * are the strains isolated in this study; ** are isolated from Vietnam 4.3 Results of virulence survey of DHAV-3 on duck embryos and ducklings 4.3.1 Results of virulence and pathogenicity of DHAV-3 on duck embryos 4.3.1.1 Results of quanitification of lethal dose 50% of duck embryos Results of the lethal dose 50% of DHAV-3 virus on duck embryos were shown in 0.2 ml of suspension containing 108.17 LD50 (ELD 50 = 10 8.17/ 0.2 ml) 4.3.1.2 Results of average embryo deaths by concentration Table 4.3 Average embryo death time according to the concentration of virus fluid Embryos die by time (hours) Dilutio Average downtime (hours) Death rate (%)