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:
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.

1


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

1


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 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.

2


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
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

3


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
]
2

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

4

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 <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
Data processing
Analyze results
3.4.2.2 Methods of analyzing the HA gene sequence of type A
H5N1 avian influenza virus
To determine the variation of type A H5N1 avian influenza virus
that causing disease and circulating in poultry, after the results of the
HA gene sequence using Molecular Evolutionary Genetics Analysis
software (MEGA 6.0) analyzed to confirm determine the level of
nucleotide variation in the HA gene segment of type A H5N1 avian
influenza virus.

5


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.

6


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
2014, 2015 and 2016.
Table 4.1: Epidemiological information on the number of poultry flocks
infected with type A H5N1 in some Mekong Delta provinces by species
Species

Chicken
Duck
MD
Total

2014
No of

Ratio
flock
(%)
32
43.2a
40
54.1a
2
2.7b
74

2015
No of Ratio
flock
(%)
18 66.7a
8 29.6b
1
3.7c
27

2016
No of Ratio
flock
(%)
14
82.3a
3
17.7b
0

0.0
17

Total
64 (54.2)
51 (43.2)
3 (2.6)
118

The values of the latex digits in the same column are not different statistically
significant (P>0,05). MD: Muscovy duck

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.

7


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

Age
group
(month)
<1
1-3
>3
Total

2014
No of Ratio
flock
(%)
1
3.1b
23 71.9a
8 25.0c
32

2015
No of Ratio
flock
(%)
1
5.6b
13
72.2a
4 22.2b
18

2016

No of Ratio
flock
(%)
0
0.0
12
85.7a
2 14.3b
14

Total
2
48
14
64

Ratio
(%)
3.1
75.0
21.9

The values of the latex digits in the same column are not different statistically
significant (P>0,05)

Table 4.2b: The morbidity rate of type A H5N1 avian influenza on
ducks in provinces of the Mekong Delta in 2014, 2015 and 2016 by age
Age
group
(month)

<1
1-3
>3
Total

2014
No of Ratio
flock
(%)
6 14.3b
35 83.3a
1
2.4c
42

2015
No of Ratio
flock
(%)
2 22.2a,c
6
66.7a
1 11.1b,c
9

2016
No of Ratio
flock
(%)
0

0.0
3 100.0
0
0.0
3

Total
8
44
2
54

Ratio
(%)
14.8
81.5
3.7

The values of the latex digits in the same column are not different statistically
significant (P>0,05)

The results investigation from 2014 to 2016 show, the number of
poultry from 1-3 months of age has the highest incidence rate of 75.0%
on chickens and 81.5% on ducks; for other age groups, there is a
difference between chickens and ducks as: on ducks with age group <1
month, the rate (14.8%) is higher than that of the age group > 3 months
(3.7%) but on chickens was opposite the age group > 3 months (21.9%)
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.

8


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
Vaccination
status
1 shot
Unvaccinated
2 shot
Total

2014
No of Ratio
flock
(%)
40 54.1a
34 45.9a
0
0.0
74

2015
No of Ratio
flock
(%)
13 48.1b
14 51.9b

0
0.0
27

2016
No of Ratio
flock
(%)
9 52.9c
8 47.1c
0
0.0
17

Total
62
56
0
118

Ratio
(%)
52.5
47.5
0.0

The values of the latex digits in the same column are not different statistically
significant (P<0,05)

The flocks of poultry infected with type A H5N1 over the years

from 2014, 2015 to 2016 are all in the group that has not been
vaccinated or has only received one injection and the incidence of
influenza in these flocks is not the difference between flocks that are
vaccinated with only one dose and unvaccinated (Table 4.3). On the
flocks of poultry that have been vaccinated with 2 shot in accordance
with the regulations, it has been well protected, there has been no
infected in all 3 years of survey. This result shows that the vaccination
procedures and vaccine used in the period of 2014 to 2016 have met and
protected poultry flocks against the pathogenicity of type A H5N1avian
influenza virus.
Table 4.4: The morbidity rate of type A H5N1 avian influenza on poultry
flocks in Mekong Delta provinces by the time
Time
(quarter)
I
II
III
IV
Total

2014
No of
Ratio
flock
(%)
54
73.0a
13
17.5b
3

4.1c
4
5.4c
74

2015
No of
Ratio
flock
(%)
13
48.2a
2
7.4b
11
40.7a
1
3.7b
27

2016
No of
Ratio
flock
(%)
8
47.1a
6 35.3a,b
3
17.6b

0
0.0
17

Total
75
21
17
5
118

Ratio
(%)
63.6a
17.8b
14.4b
4.2c

The values of the latex digits in the same column are not different statistically
significant (P<0,05)

9


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
Population
(bird)
< 200
200 – 500
>500 – 1.000
>1.000

Total

2014
No of
Ratio
flock
(%)
13
17.5a
14
19.0a
19 25.7a,b
28
37.8b
74

2015
No of

Ratio
flock
(%)
5
18.6a
4
14.8a
11
40.7b
7 25.9a,b
27

2016
No of
Ratio
flock
(%)
1
5.9a
7
41.2b
5 29.4a,b
4 23.5a,b
17

Total
19
25
35
39

118

Ratio
(%)
16.1
21.2
29.7
33.0

The values of the latex digits in the same column are not different statistically
significant (P<0,05)

In 2014, the proportion of flocks of poultry infected with
influenza increased according to herd size, the difference between the
number of flocks of over 1000 heads compared to flocks of less than
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.

10


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
3

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
9
16.7
11
20.4
21
38.9
14
21.9
22
40.7
1
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
2
3.1
1
1.9
Thigh muscle hemorrhages
9
14.1
4

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
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
Thick air sac
5
7.8
4
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 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.

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
7
6
3
0
0

0
4
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

2
5
11
2
0
2
4
2
2
5
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

3
0
10
2
9
1
4
7
0
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

5
12
27
7
9
3
8
13
2
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<0,05)

Table 4.9: prevalence rate of type A H5N1 avian influenza virus on health
poultry sold in markets, slaughterhouses or poultry farms
Number of positive
Ratio
Ratio
N1
H5N1
(%)
(%)
15.1
90
9.6
90

Location

No

Markets

938

142

Slaughterhouses

236

21

8.9

12

5.1

12

5.1b

Farms

182

0

0.0

0

0.0

0

0.0

H5

Ratio
(%)
9.6a

The values of the latex digits in the same column are not different statistically significant
(P<0,05). No: number of samples

Investigation circulation results of type A H5N1 avian influenza
virus showed that the circulation of influenza virus was not detected in
poultry flocks in households. The prevalence of influenza virus in the
market was 9.6% higher than that of the slaughterhouse was 5.1% and
this difference is statistically significant.

13


Table 4.10: The prevalence rate of type A H5N1 avian influenza virus
period 2014-2016 by species
Provinces,
city

No

An Giang
Bac Lieu
Ca Mau
Can Tho
Đong Thap

Hau Giang
Kien Giang
Soc Trang
Tra Vinh
Vinh Long
Total

54
30
30
54
54
24
54
24
42
54
420

Chicken
Ratio
NoP
(%)
3
5.5
0
0.0
2
6.7
2

3.7
0
0.0
1
4.2
5
9.3
0
0.0
1
2.4
3
5.5
17
4.0a

Duck
No

NoP

84
102
102
84
84
114
84
114
84

84
936

2
12
25
5
9
2
3
13
1
13
85

Total

Ratio
(%)
2.4
11.8
24.5
5.9
10.7
1.7
3.6
11.4
1.2
15.5
9.1b

No

NoP

138
132
132
138
138
138
138
138
126
138
1.356

5
12
27
7
9
3
8
13
2
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<0,05)

Investigation circulation results of type A H5N1 avian influenza
virus by species showed that the prevalence of influenza virus on chickens
only detected in 7/10 provinces and cities surveyed, accounting for 4.0%
lower compared to ducks at 10/10 provinces, the survey ratio was 9.1% and
this difference was statistically significant. This result is consistent with
Sturm Ramirez et al. (2005), ducks are carriers of type A H5N1 avian
influenza virus in the study of the role of ducks in avian influenza H5N1 in
Asia.
4.2 Result of 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.
4.2.1 Result of sequencing 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.
From 2014 to 2016, a total of 49 type A H5N1 avian influenza
strains have been selected to sequence nucleotide of the HA gene segment,
including 25 circulating virus strains, 23 causing disease strains in poultry

14


and 1 viral strain exists in the enviroment (land) of 10 provinces and cities
surveyed in the Mekong Delta.
The sequences of gene HA of type A H5N1 avian influenza virus is
from 1,615 to 1,717 nucleotides.
4.2.2 Result of 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.
4.2.2.1 Compare in the same year and between years
The results compared the different nucleotide of HA gene segments
of type A H5N1 influenza virus strains in the same year 2014, 2015 and
between 2014 and 2015 with the lowest number of nucleotides is 0
nucleotide and the highest is 91 nucleotides are equivalent to the difference
rate of 0.0–5.7%.
The number of different nucleotides between HA gene segment of
type A H5N1 avian influenza viruses in the same year 2014, 2016 and
between 2014 and 2016 was from 2-151 nucleotides equivalent to the
difference rate of 0.1% - 9.5%. This rate is higher than the difference rate in
the same period of 2014, 2015 and between 2014 and 2015 with the rate
from 0.0 to 5.7%.

The results compared the sequence of nucleotides on the HA gene
segment of the H5N1 avian influenza virus that causes disease and
circulates in poultry in the Mekong Delta provinces in the same year 2015,
2016 and between 2015 and 2016 was from 2–151 nucleotides are
equivalent to the difference rate of 0.1% - 9.5%.
4.2.2.2 Compare with published strains in Vietnam and in the world
When comparing HA gene segments nucleotide sequences of type A
H5N1 influenza virus strains causing disease in poultry in 2014 with strains
published in Vietnam and some countries in the world, it shows that there
was a difference rate from 1, 2-10.2%.
Similarly, when comparing 11 strains of type A H5N1 avian
influenza that represent the sequence of sequencing in 2015, there was a
difference rate from 1.0-12.4% compared to 8 strains of virus published in
Vietnam and 6 strains in the world and 11 types of H5N1 avian influenza
strains sequenced in 2016 have a different rate of nucleotide than those
published in Vietnam and in the world from 0.3 -13.2%.
4.2.2.3 Comparison between causing disease and circulating
The nucleotide level difference between H5N1 avian influenza viruses that
causing disease and circulating in poultry in 10 provinces and cities in the

15


Mekong Delta in 2015 ranges from 6 to 91 nucleotides, accounting for a
rate of 0.4– 5.7%, corresponding to the proportion of 94.3–99.6%.
In 2016, the nucleotide level difference between causing disease and
circulating avian influenza A H5N1 strains ranged from 8–142 nucleotides,
accounting for 0.5–8.9%; The corresponding rate is from 91.1 to 99.5%.
4.2.2.4 Comparison between causing disease and circulating together
The strains of type A H5N1 influenza virus causing disease in

poultry in 2014 and 2015 have a different number of nucleotides to the
causing disease viruses in 2016 from 14-82 nucleotides, accounting for the
rate of 0.9-5.1. % corresponds to the similarity rate of 94.9–99.1%.
Comparison results between strains circulating in 2015 and 2016
showed that the circulating strains in the same locality or neighboring
provinces will have very high similarity (99.8-99.9 %) in other words, the
same subtype of type A H5N1 bird flu virus.
4.2.2.5 Compare with the viruses strains infected on human
The sequence of sequencing viruses in 2015 has a high similarity rate
compared to A/Vietnam/CM32/ 2011 (H5N1) strains causing human
diseases in Ca Mau in 2011 with the number of differences from 62-75
nucleotides, corresponding to the difference rate of 3.8-4.7% and had the
lowest similarity rate compared to A/Vietnam/UT36236/2010 (H5N1)
strains causing human disease in Vietnam in 2010 with the number
different from 141-151 nucleotides, corresponding to the difference rate of
8.7-9.4%.
4.3 Result of 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.
To determine the subclade of type A H5N1 avian influenza virus using
MEGA 6.0 software to analyze and build a phylogenetic tree. In addition to
the virus strains that have been sequenced in this study (3 strains in 2014,
24 strains in 2015 and 22 strains in 2016), we use the sequence of 32
reference virus strains and 7 strains of virus that causing disease on humans
to build the phylogenetic tree, the results are shown in Figure 4.1-4.3.
The results of building the phylogenetic tree determined that the H5N1
avian influenza virus strains isolated on poultry from 2014 - 2016 belong to
the viral subclade 2.3.2.1d because they are still in the same branch with
the viral strain with virus strains belonging to subclade 2.3.2.1d.

16


: Poultry viral strains in 2014
: Reference viral strains
: Infected human viral strains

Figure 4.1: Phylogenetic tree of type A H5N1 avian influenza virus strains
in 2014
Phylogenetic tree are built using MEGA 6.0 software (Tamura et al. 2013) with the
Neighbor-Joining method and Bootstap reliability factor 1000 times. Nomenclature of virus
subtypes based on WHO / OIE / FAO criteria (2014)

17


: Poultry viral strains in 2015
: Reference viral strains
: Infected human viral strains

Figure 4.2: Phylogenetic tree of type A H5N1 avian influenza virus strains
in 2015
Phylogenetic tree are built using MEGA 6.0 software (Tamura et al. 2013) with the
Neighbor-Joining method and Bootstap reliability factor 1000 times. Nomenclature of virus
subtypes based on WHO / OIE / FAO criteria (2014)

18

: Poultry viral strains in 2016
: Reference viral strains
: Infected human viral strains

Figure 4.3: Phylogenetic tree of type A H5N1 avian influenza virus strains
in 2016
Phylogenetic tree are built using MEGA 6.0 software (Tamura et al. 2013) with the
Neighbor-Joining method and Bootstap reliability factor 1000 times. Nomenclature of virus
subtypes based on WHO / OIE / FAO criteria (2014)

19


4.4 Result of analysing on HA protein amino acid sequence to
determine pathogenicity of type A H5N1 avian influenza virus.
4.4.1 4.4 Result of analysing on HA protein amino acid sequence
at cleavage site (HA0) of type A H5N1 avian influenza virus in 2014,
2015 and 2016.
The results of analysis the amino acid sequence at the cleavage site
(HA0) of avian influenza viruses in 2014, 2015 and 2016 showed that the
sequence of amino acids from positions 341 to 346 are sequenced RRR - K
R.
4.4.2 Result of analysing on HA protein amino acid sequence to
determine pathogenicity of type A H5N1 avian influenza virus in 2014,
2015 and 2016.
Table 4.11 Result of analysing on HA protein amino acid sequence to
determine pathogenicity of type A H5N1 virus in 2014
Type A H5N1 avian
influenza virus strains
A/Chick/VL/1483/2014

A/Chick/TV/1484/2014
A/Duck/TV/1485/2014

The variable position on the HA protein increases the
ability to bind to human α 2-6 receptor of type A
H5N1 avian influenza virus
149
171 171N 205 251P
513
110N
A
N
172A
R
K
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+

+
+
+
+
+
0

“+”: variable; 0: not variable

The strains of type A H5N1 influenza virus causing disease in
poultry in 2014 had changes in specific positions including 110N, 149A,
171N, 172A, 205R, 251P and no change at position 513K. According to Su
et al. (2008) when there was a change in position 110N, it would increase
the ability to bind to α 2-6 receptors to bind to human cell. Besides, Yang et
al. (2007) reported that the occurrence of variation in position 149A
increased the ability to bind to the α 2-6 receptor binding to human cells. In
addition, Wang et al. (2010) suggested that variation in positions 171N,
172A, 205R; Watanabe et al. (2011) indicated that there were change at
251P and Yamada et al. (2006) when modified at position 513K also
increased the ability to bind to α 2-6 receptor attached to human cell.

20


Thus, the presence of variable sites on HA protein including 110N, 149A,
171N, 172A, 205R, 251P will be risk factors to increase the ability to bind
to the α 2-6 receptors attached to the human cell of type A H5N1 influenza
virus strains causing disease in poultry in 2014.
Table 4.12 Result of analysing on HA protein amino acid sequence to
determine pathogenicity of type A H5N1 virus in 2015

Type A H5N1 avian
influenza virus strains
A/Duck/CM/1502/2015
A/Chick/CT/1503/2015
A/Chick/CT/1504/2015
A/Chick/KG/1505/2015
A/Duck/CM/1506/2015
A/Chick/CM/1508/2015
A/Duck/CM/1509/2015
A/Duck/VL/1515/2015
A/Duck/VL/7/2015
A/Chick/KG/26/2015
A/Chick/KG/34/2015
A/Chick/VL/178/2015
A/Duck/VL/186/2015
A/Duck/VL/189/2015
A/Chick/HG/210/2015
A/Duck/ST/259/2015
A/Chick/VL/329/2015
A/Duck/AG/676/2015
A/Duck/AG/677/2015
A/Duck/CM/1389/2015
A/Duck/CM/3311/2015
A/Duck/BL/4633/2015
A/Duck/BL/4696/2015
A/Duck/BL/4749/2015

The variable position on the HA protein increases the
ability to bind to human α 2-6 receptor of type A
H5N1 avian influenza virus

110 149 171
171N;
205 251 513
N
A
N
172A
R
P
K
+
+
+
+
+
+
0
+
+
0
0
+
+
0
+
+
+
+
+
+

0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+

+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+

+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+

+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0

+
+
+
+
0
0
0
+
+
+
+
0
+
0
+
+
+
+
+
+
0
+
+
+
+
0
+
0

“+”: variable; 0: not variable

21


The strains of H5N1 influenza virus causing disease and circulating
on poultry in 2015 have changed in specific positions including 110N,
149A, 171N, 172A, 205R, 251P and no change at position 513K. However,
at the amino acid position 171 of A/Chick/CT/1503/2015, it is not
Asparagin (N) but has been converted to aspartic acid (D). Similarly, as the
virus strains in 2014, the presence of variable positions on HA protein
include 110N, 149A, 171N, 172A, 205R, 251P will be risk factors to
increase the ability to bind to the α 2-6 receptors attached to the human cell
of type A H5N1 influenza virus.
Table 4.13 Result of analysing on HA protein amino acid sequence to
determine pathogenicity of type A H5N1 virus in 2016
Type A H5N1 avian influenza
virus strains
A/Chick/CT/1603/2016
A/Duck/CT/1604/2016
A/Duck/TV/1605/2016
A/Duck/CT/1606/2016
A/Chick/ST/1607/2016
A/Chick/ST/1608/2016
A/Chick/CT/1613/2016
A/Chick/CT/1617/2016
A/Chick/KG/1619/2016
A/Chick/ST/1625/2016
A/Chick/KG/1627/2016
A/Chick/CM/1635/2016
A/Chick/AG/0010/2016

A/Duck/CM/0057/2016
A/Chick/CM/0062/2016
A/Muscovy duck/CM/852/2016
A/Enviroment/CM/1662/2016
A/Duck/DT/1760/2016
A/Duck/DT/1763/2016
A/Duck/DT/1767/2016
A/Muscovy duck/CM/1834/2016
A/Chick/CM/1846/2016

The variable position on the HA protein increases the
ability to bind to human α 2-6 receptor of type A
H5N1 avian influenza virus
110 149 171
171N; 205 251
513K
N
A
N
172A
R
P
+
+
+
+
+
+
0
+

+
+
+
+
+
0
+
+
0
0
+
+
0
+
+
+
+
+
+
0
+
+
+
+
0
+
0
+
+
+

+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
0
0
+
+
+
+
0
+
0
+
+
+
+
+

+
0
+
+
+
+
+
0
0
+
+
+
+
+
+
0
+
+
0
0
+
+
0
+
+
+
+
+
+
0

+
+
+
+
0
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+
+
+
+
+
0
+
+

+
+
+
+
0
+
+
+
+
+
+
0
+
+
0
0
+
+
+
+
+
+
+
+
+
0

“+”: variable; 0: not variable

22