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

Circulation and genetic variation of type A H5N1 avian influenza virus on poultry in some Mekong river delta provinces.. Circulation and genetic variation of type A/H5N1 avian influenza

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:

1 Learning Resource Center, Can Tho University

2 National Library of VietNam

PUBLISHED ARTICLES

1 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

2 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

3 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 1 (2019): 36-41

4 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

5 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

- 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 2014-

2016 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 1

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 2

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 3

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 4

Analysing of amino acid sequence on HA protein to determine

pathogenicity of type A H5N1 avian influenza virus

3.2 Timeline and researching areas

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

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 - ]

n: number of samples to take

p1: probability to detect disease (0,95)

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 <30 cycles) to perform the sequence of HA gene of type A H5N1 avian influenza virus

Steps to implement the gene sequencing process

Prepare sequence samples

Implementation of the HA amplification cycle

Prepare and perform the sequencing cycle

3.4.3 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

After acquiring HA gene sequences of the type A H5N1 avian influenza virus, a phylogenetic tree will be constructed using Molecular Evolutionary Genetics Analysis (MEGA 6.0)

Criteria to determine the type A H5N1 avian influenza virus subclade:

Based on standardized classification criteria applied by WHO/OIE/FAO (2009, 2012, 2014), the determination of the new subclade of type A H5N1 influenza virus must be based on criteria including: a group of HA gene sequences of type A H5N1 avian influenza virus (from 4 or more sequences) with bootstrap values from

60 to above and the proportion of nucleotide component differences must be greater than 1.5% when compared to categorized subclade groups

3.4.4 Methods of analysing of amino acid sequence on HA protein to determine pathogenicity of type A H5N1 avian influenza virus

After having the HA genes sequence of type A H5N1 avian influenza virus, using Molecular Evoluationary Genetics Analysis software (MEGA 6.0) converted to amino acid sequence of HA protein

to conduct analysis and comparison the differences of amino acid at sites that regulate host cell binding on the HA protein to determine the ability to bind to host cells of the type A H5N1 avian influenza virus through the receptor on H antigen Since then, the ability to cause disease in poultry or humans of type A H5N1 avian influenza virus has been determined

3.4.5 Method of calculating and data processing

Data in the study were processed by software Minitab 19.0, Chi-square

test, Chi-square yates test

CHAPTER 4: RESULTS AND DISCUSIONS 4.1 Result of epidemiological information investigation, 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

4.1.1 Result of epidemiological information investigation, clinical signs, lesions of type A H5N1 avian influenza disease in

No of flock

Ratio (%)

No of flock

Ratio (%)

In 2014, the largest number of infected poultry flocks included

32 chickens flocks, 40 ducks flocks and 2 muscovy duck flocks In

2015, there were only 18 chickens flocks, 8 ducks flocks and 1 muscovy duck flocks were infected In 2016 the number of infected poultry decreased to 14 flocks of chickens and 3 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 4.2a: The morbidity rate of type A H5N1 avian influenza on chickens in provinces of the Mekong Delta in 2014, 2015 and 2016 by age

No of flock

Ratio (%)

No of flock

Ratio (%)

No of flock

Ratio (%)

No of flock

Ratio (%)

is higher than age group < 1 month (3.1%) The difference in the prevalence of avian influenza over year, the ages of 1-3 months of age compared to the other groups is statistically significant

Table 4.3: The morbidity rate of type A H5N1 avian influenza on poultry flocks in provinces of the Mekong Delta according to vaccination status

Ratio (%)

No of flock

Ratio (%)

No of flock

Ratio (%)

Table 4.4: The morbidity rate of type A H5N1 avian influenza on poultry flocks in Mekong Delta provinces by the time

No of flock

Ratio (%)

No of flock

Ratio (%)

Investigation results of type A H5N1 avian influenza occurred in Mekong Delta provinces in the period of 2014 to 2016 showed, from January to March (first quarter) is the period of occurrence of the highest bird flu (75 flocks), accounting for 63.6%, Compared to the rest

of the year, from the second quarter to the fourth quarter, the disease appears only sporadically in some flocks of poultry (from 5 to 21 groups), accounting for 4.2 to 17.4% The difference between the total number of infected poultry in the first quarter and the remaining quarters in the period 2014-2016 is statistically significant

Table 4.5: The morbidity rate of type A H5N1 avian influenza in poultry flocks in Mekong Delta provinces by population

No of flock

Ratio (%)

No of flock

Ratio (%)

200 heads and from 200-500 birds were statistical significance However, in 2015 and 2016 the number of flocks of poultry with a flock size of more than 1000 heads was not statistically significant compared

to the other herd sizes

Table 4.6: Frequency of clinical signs of type A H5N1 avian influenza disease (n= 118)

Clinical signs

Chicken (n=64) Duck, muscovy duck

(n=54) Frequency Ratio

Table 4.7: Frequency of lesions of type A H5N1 avian influenza disease (n= 118)

Lesions

Chicken (n=64) Duck, muscovy

duck (n=54) Frequency Ratio

(%)

Frequency Ratio

(%) Chest muscles hemorrhages 2 3.1 1 1.9 Thigh muscle hemorrhages 9 14.1 4 7.4 Trachea hemorrhages, fluid 11 17.2 10 18.5

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 9 16.7 Proventriculus hemorrhages 9 14.1 6 11.1 Intestine hemorrhages 11 17.2 7 12.9 Fabricius swelling, hemorrhages 11 17.2 7 12.9

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 5 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 9 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