KHẢO sát đặc TÍNH gây BỆNH và BIẾN đổi DI TRUYỀN của VIRUS cúm GIA cầm TYPE a h5n1 lưu HÀNH tại ĐỒNG BẰNG SÔNG cửu LONG GIAI đoạn 2014 2016 tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: PATHOLOGY AND TREATMENT OF ANIMALS Major code: 62 64 01 02 TIEN NGOC TIEN INVESTIGATING THE PATHOGENICITY AND GENETIC VARIATION OF TYPE A H5N1 AVIAN INFLUENZA VIRUS CIRCULATED IN MEKONG DELTA IN THE PERIOD 2014-2016 Can Tho, 2020 THIS THESIS WAS COMPLETED AT CAN THO UNIVERSITY Academic supervisor: Assoc Prof.DR Ly Thi Lien Khai This thesis was defended against the Ph.D dissertation council at the university level Place: ………… Can Tho University Time: At ……., …………………… 1st Opponent: 2nd Opponent: 3rd Opponent: Thesis could be found at: Learning Resource Center, Can Tho University National Library of VietNam PUBLISHED ARTICLES Tien Ngoc Tien, Quach Thuy Lan, Nguyen Khoa and Ly Thi Lien Khai, 2016 Circulation and genetic variation of type A H5N1 avian influenza virus on poultry in some Mekong river delta provinces Can Tho University Journal of Science., Special issue agriculture, pp 142-151 Tien Ngoc Tien, Phung Thi Thanh Thuy, Nguyen Quoc Hung, Tran Dien Quy and Ly Thi Lien Khai, 2017 Circulation and genetic variation of type A/H5N1 avian influenza virus on poultry in some Mekong river delta provinces Veterinary sciences and Techniques, No 3, 2017, pp 5-13 Tien Ngoc Tien and Ly Thi Lien Khai, 2019 Comparision of genetic variation between avian influenza type A H5N1 virus causing disease and circulating on poultry in some provinces in the Mekong Delta in 2016 Can Tho University Journal of Science Vol 11, No (2019): 36-41 Tien Ngoc Tien, Truong Thi Kim Dung, Nguyen Anh Dung, Nguyen Thanh Huy, Ly Thi Lien Khai, 2015 Investigation on the circulation of high pathogenic type A H5N1 avian influenza virus on ducks and poultry in enclosed environment in Ca Mau provine and Can Tho city in 2014 Proceedings of National conference on Animal Veterinary sciences, 2015 Tien Ngoc Tien, Ly Thi Lien Khai, 2017 Circulation and genetic variation of type A H5N1 avian influenza virus on poultry in An Giang, Kien Giang, Ca Mau and Can Tho city in 2016 Proceedings of National conference on Animal Veterinary sciences, 2017 Chapter 1: INTRODUCTION Avian influenza disease has appeared in Vietnam since 2003 in some provinces such as Ha Tay, Tien Giang and Long An and then spread to most provinces and cities throughout the country, killing and destroying millions of poultry, causing great damage to the poultry industry of our country In recent years, according to the results of monitoring the genetic variation of the type A H5N1 avian influenza virus of the Department of Animal Health, the virus has changed constantly to create different virus subclades (Department of Animal Health, 2015) Especially, in the previous years in some provinces in the Mekong Delta such as Dong Thap, Kien Giang and Soc Trang, cases of H5N1 avian influenza have been continuously reported on humans and fatal with a high rate With fast spreading characteristics, circulating on healthy poultry flocks, new virus subclades that are capable of altering pathogens with high mortality rates of human type A H5N1 avian influenza are detected, early detection, control of outbreaks, destruction of infected poultry, Determining the pathogenicity and genetic variation of type A H5N1 avian influenza virus is very urgent and very important Therefore, the study "Investigating the pathogenicity and genetic variation of A H5N1 type avian influenza virus circulating in the Mekong Delta in the period 2014-2016" was conducted Objectives - To identify epidemiological characteristics, clinical signs, lesions of type A H5N1 avian influenza disease and prevalence of type A H5N1 avian influenza virus in healthy poultry flocks raised in livestock households, traded in markets and in poultry slaughterhouses - To determine the genetic variation of type A H5N1 avian influenza virus that causing disease and circulating in the Mekong Delta provinces - To determine the genetic correlation between type A H5N1 avian influenza viruses that causing disease and circulating in poultry; between type A H5N1 avian influenza viruses detected in the study and type A H5N1 influenza viruses have been published in Vietnam and the world - To determine of pathogenicity of type A H5N1 avian influenza viruses in the Mekong Delta provinces based on the amino acid sequence of the HA protein Significance This is the study of the epidemiological situation of type A H5N1 avian influenza disease and the genetic variation of type A H5N1 avian influenza virus, is a scientific basis for the implementation of effective measures to prevent and control diseases, helping poultry industry to develop and contribute to protecting human health Innovative contributions of the thesis - Identification the subclade of type A H5N1 avian influenza virus circulating and causing disease in poultry during the period 20142016 in the Mekong Delta provinces belong to subclade 2.3.2.1d - Determination of changes in amino acid positions on HA protein is a risk factor that increases the ability to bind to α 2-6 receptors that bind to cells and cause disease in human - A number of amino acid positions 82, 152, 185, 282 have been identified that play an important role in the pathogenicity of type A H5N1 avian influenza virus, the variation of amino acids in these locations will alter the ability to cause disease, the type A H5N1 avian influenza virus can convert the pathogen from poultry to human disease and vice versa Chapter 3: RESEARCH METHODOLOGY 3.1 Content 3.1.1 Content Collecting epidemiological information, clinical signs, lesions, testing and development of an epidemiological map of type A H5N1 avian influenza disease and investigation the circulation of type A H5N1 avian influenza virus in some provinces in the Mekong Delta 3.1.2 Content Sequencing and analyzing the HA genes sequence of type A H5N1 avian influenza virus causing disease and circulating on healthy poultry flocks raised in livestock households, traded in markets and poultry slaughterhouses 3.1.3 Content Building phylogenetic tree from the type A H5N1 avian influenza virus that causing disease and circulating in poultry found in the study and type A H5N1 influenza viruses have been published in Vietnam and the world 3.1.4 Content Analysing of amino acid sequence on HA protein to determine pathogenicity of type A H5N1 avian influenza virus 3.2 Timeline and researching areas 3.2.1 Timeline The thesis is carried out from August, 2014 to December, 2018 3.2.2 Researching areas The thesis was conducted at Can Tho University Regional Animal Health Ofice No VII An Giang, Bac Lieu, Ca Mau, Dong Thap, Hau Giang, Kien Giang, Soc Trang, Tra Vinh, Vinh Long and Can Tho city Gene sequencing: Macrogen company, South Korea 3.4 Research methology 3.4.1 Methods of collecting epidemiological information, clinical signs, lesions, testing, development of an epidemiological map of type A H5N1 avian influenza disease and investigation the circulation of type A H5N1 avian influenza virus in some provinces in the Mekong Delta 3.4.1.1 Methods of collecting epidemiological information, clinical signs, lesions, testing, development of an epidemiological map of type A H5N1 avian influenza disease When detecting poultry flocks (chickens, ducks, muscovy ducks) had some suspected clinical sign of avian influenza disease, we recorded epidemiological information according to the form in Appendix 1, then examination performed the lesions and collected samples to test the type A H5N1 avian influenza by Real time RT-PCR method and development of an epidemiological map to determine the spatial, temporal and confidential distribution by Quantum Gis software 3.4.1.2 Methods of investigating the circulation of type A H5N1 avian influenza virus in some provinces in the Mekong Delta To investigate the prevalence of type A H5N1 avian influenza virus in poultry sold in markets, slaughterhouses or poultry farms, we took swab samples to test for avian influenza virus The sample capacity to be taken is calculated by the following formula: n = [1 - (1 - p1)1/d] x [N - d-1 ] n: number of samples to take p1: probability to detect disease (0,95) d: number of infected (d = N x p2) p2: prevalence rate predicted N: population Table 3.1: Distribution of samples and number of swab samples taken over the years in provinces and city Provinces, city An Giang Bac Lieu Ca Mau Can Tho Đong Thap Hau Giang Kien Giang Soc Trang Tra Vinh Vinh Long Total Number of samples 2014 2015 36 42 36 36 36 36 36 42 36 42 36 42 36 42 36 42 36 42 36 42 360 408 2016 60 60 60 60 60 60 60 60 48 60 588 Total 138 132 132 138 138 138 138 138 126 138 1,356 3.4.1.3 Methods of testing for A H5N1 avian influenza virus and swab samples (Real time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR)) Process of real time RT-PCR reaction (Department of Animal Health, 2009, 2014) 3.4.2 Methods of sequencing and analyzing the HA genes sequence of type A H5N1 avian influenza virus 3.4.2.1 Methods of sequencing the HA genes sequence of type A H5N1 avian influenza virus After identifying positive test samples of A H5N1 avian influenza virus by Real time RT-PCR method, 49 representative samples were selected for poultry species, representing localities and satisfactory testing by sequencing method (with threshold 0,05) MD: Muscovy duck In 2014, the largest number of infected poultry flocks included 32 chickens flocks, 40 ducks flocks and muscovy duck flocks In 2015, there were only 18 chickens flocks, ducks flocks and muscovy duck flocks were infected In 2016 the number of infected poultry decreased to 14 flocks of chickens and ducks The results of comparing the difference of the number of infected poultry flocks over the years show that in 2014 the number of ducks and chickens infected with influenza is high and higher than muscovy duck and this difference is statistically significant However, in 2015 and 2016 the number of chickens is more infected than ducks and muscovy duck and this difference is statistically significant with the value of P = 0.000 Table 4.6: Frequency of clinical signs of type A H5N1 avian influenza disease (n= 118) Chicken (n=64) Clinical signs Depression Anorexia Edema of the head Respiratory signs Edematous comb Leg paralysis Wings paralysis Green to white diarrhea Shanks hemorrhages Neurological sign Frequency 64 64 13 14 15 11 10 27 15 Ratio (%) 100.0 100.0 20.3 21.9 23.4 17.2 15.6 42.2 23.4 4.7 Duck, muscovy duck (n=54) Frequency Ratio (%) 54 100.0 54 100.0 16.7 11 20.4 21 38.9 14 21.9 22 40.7 1.8 22 40.7 Poultry flocks infected with the highly pathogenic type A H5N1 influenza virus exhibit common symptoms such as depression, anorexia (100%), edema of the head, respiratory signs, leg paralysis, wings paralysis, green to white diarrhea, shanks hemorrhages; neurological sign Shanks hemorrhages found in chickens (23.4%) with higher frequency than ducks (1.8%) are characteristic manifestations of differential diagnosis of type A H5N1 avian influenza disease Neurological sign appear more in ducks, muscovy ducks (40.7%) than chickens (4.7%) and are also typical symptoms for clinical diagnosis of type A H5N1 avian influenza disease in ducks 11 Table 4.7: Frequency of lesions of type A H5N1 avian influenza disease (n= 118) Chicken (n=64) Duck, muscovy duck (n=54) Lesions Frequency Ratio Frequency Ratio (%) (%) Chest muscles hemorrhages 3.1 1.9 Thigh muscle hemorrhages 14.1 7.4 Trachea hemorrhages, fluid 11 17.2 10 18.5 Lungs hemorrhages 24 37.5 15 27.8 Hemorrhages on heart perirenal 22 34.4 14 25.9 Liver swelling, hemorrhages 23 35.9 15 27.8 Spleen swelling, hemorrhages 23 35.9 13 24.1 Kidney swelling, hemorrhages 17 26.6 16.7 Proventriculus hemorrhages 14.1 11.1 Intestine hemorrhages 11 17.2 12.9 Fabricius swelling, hemorrhages 11 17.2 12.9 Thick air sac 7.8 7.4 Brain hemorrhages 40 62.5 31 57.4 Poultry flocks infected with the highly pathogenic type A H5N1 influenza virus have lesion such as brain hemorrhages; lungs hemorrhages; hemorrhages on heart perirenal; liver swelling, hemorrhages; spleen swelling, hemorrhages; trachea hemorrhages are lesions that appear quite high on chickens and lower on ducks (Table 4.8) 4.1.4 The circulation of type A H5N1 avian influenza virus in some provinces in the Mekong Delta The results show that an average circulation rate of type A H5N1 avian influenza virus was 7.5% In 2014, there was the circulation of type A H5N1 avian influenza virus in localities surveyed with an average circulation rate was 6.7% In 2015, the number of localities circulating the type A H5N1 avian influenza virus increased to provinces and cities with an average circulation rate was 8.6%, higher than in 2014 However, this difference is not statistically significant (p> 0.05) Survey results of the circulation of type A H5N1 avian influenza virus in 2016 with the average prevalence decreased to only 7.3% lower than in 2015 but this difference is not significant (p> 0.05) Detailed results are shown in Table 4.8 12 Table 4.8: The prevalence rate of type A H5N1 avian influenza virus on health poultry in 2014, 2015 and 2016 2014 Provinces, city No NoP An Giang Bac Lieu Ca Mau Can Tho Đong Thap Hau Giang Kien Giang Soc Trang Tra Vinh Vinh Long Total 36 36 36 36 36 36 36 36 36 36 360 0 4 24 2015 Ratio (%) 0.0 19.4 16.7 8.3 0.0 0.0 0.0 11.1 0.0 11.1 6.7a No NoP 42 36 36 42 42 42 42 42 42 42 408 11 2 2 35 2016 Ratio (%) 4.8 13.9 33.3 4.8 0.0 4.8 9.4 4.8 4.8 11.9 8.6a No NoP 60 60 60 60 60 60 60 60 48 60 588 10 7 43 Total Ratio (%) 6.6 0.0 16.6 3.3 15.0 1.6 6.6 11.6 0.0 11.6 7.3a No NoP 138 132 132 138 138 138 138 138 126 138 1,356 12 27 13 16 102 Ratio (%) 3.6 9.1 20.5 5.1 6.5 2.2 5.8 9.4 1.6 11.6 7.5 No: number of samples; NoP: number of positive H5N1 The values of the latex digits in the same column are not different statistically significant (P