GENETICALLY VARIATION OF PB2, PB1 AND PA POLYMERASE GENES OF THE AH5N1 INFLUENZA VIRUS RECENTLY ISOLATED IN VIETNAM

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH
HANOI MEDICAL UNIVERSITY
NGUYEN MANH KIEN
GENETICALLY VARIATION
OF PB2, PB1 AND PA POLYMERASE GENES
OF THE A/H5N1 INFLUENZA VIRUS
RECENTLY ISOLATED IN VIETNAM
Speciality: Medical Biochemistry
Code : 62 72 01 12
SUMMARY OF THE MEDICAL DISSERTATION
HANOI – 2014
The dissertation was completed at: HANOI MEDICAL UNIVERSITY.
The scientific guidance:
1. Assoc. Prof, Dr. Le Thanh Hoa.
2. Assoc. Prof, Dr. Dang Thi Ngoc Dung.
Reviewers 1: Assoc. Prof, Dr. Bach Vong Hai
Vietnam Military Medical Academy
Reviewers 2: Prof, Dr. Dang Duc Anh
National Institute of Hygiene and Epidemiology
Reviewers 3: Assoc. Prof, Dr. Dinh Duy Khang
Institute of Biotechnology
The dissertation will have defended before the Council of protect
dissertation Hanoi Medical University.
Meeting at: The Hall protect dissertation - Hanoi Medical
University, Number 1, Ton That Tung - Dong Da - Ha Noi.
At …… hour, 2014 …. …
Can find dissertation at the library:
- Library National.
- Library Hanoi Medical University.
- Library Central Health information.
LIST OF THE AUTHOR’S

PUBLICATIONS RELATED TO THE DISSERTATION
1. Nguyen Manh Kien, Nguyen Thi Bich Nga, Le Thanh Hoa
(2008), “Properties of the HA(H5) gene for the A/H5N1 strains
of the Fujian sublineage isolated in infected poultry and human
in Vietnam in 2007”, Journal of Military Pharmaco -
medicine, 33(8), pp. 29-36.
2. Nguyen Manh Kien, Nguyen Thi Bich Nga, Doan Thi Thanh
Huong, Dang Thi Ngoc Dung, Le Thanh Hoa
(2011), “Molecular properties of polymerase PB1 gene of
DkNA72 and DkNA14 isolates of the Fujian sublineeage
A/H5N1 influenza virus isolated in Vietnam”, Journal of
Military Pharmaco - medicine, 36(1), pp. 36-41.
3. Nguyen Manh Kien, Dang Thi Ngoc Dung, Le Thanh Hoa
(2012), “Molecular properties of the polymerase complex of
the A/H5N1 clade 2.3.2.1 isolated from ducks in Quang Tri
province in 2011”, Journal of Vietnam Medicine, 396(1), pp.
50-56.

ABREVIATIONS IN THE DISSERTATION
bp base pair
Da dalton
DNA Deoxyribonucleic acid
dNTP Deoxy Nucleotide Triphosphate
ddNTP dideoxy Nucleotide Triphosphate
FAO Food and Agricultural Organization
HA Hemagglutinin
kb Kilo base
M Matrix protein
MEGA Molecular Evolutionary Genetics Analysis
NA Neuraminidase

NEP Nuclear Export Protein
NP Nucleoprotein
NS Non-structural protein
OIE Office International des Epizooties
PA Polymerase acidic protein
PB1 Polymerase basic protein 1
PB2 Polymerase basic protein 2
RT-PCR Revertranscription Polymerase Chain Reaction
RNA Ribonucleic acid
RNP Ribonucleoprotein
(-) ssRNA Negative single-strand Ribonucleic Acid
WHO World Health Organization
1
INTRODUTION
1. The reality and imperative of the topic
The A influenza virus belongs to Orthomyxoviridae family, with
many various subtype viruses circulate in the wild bird, can to
change them infected and transmition abilities to human and many
animal specieses, caused A influenza pandemics in human in history.
PB2, PB1 and PA genes encoding PB2, PB1 and PA polymerase
proteins, are subunits of enzym polymerase complex. This complex
is importal role to decide adapted transcription of the virus in the
host specieses. In addition, PB1 gen contants 2 open reading frame
(ORF), encoding PB1-F2 and PB1-N40 proteins, that join in the
virulence expresion of the A influenza virus in the infected host.
Since 2003, A/H5N1 highly virulent virus cause many A/H5N1
oubreaks in the poultry, infected and cause serious acute flu with the
high mortality rate (over 60%) in the human population. In there,
Vietnam is one of countries, that have A/H5N1 avian influenza
oubreaks in 2003 and repeated in many provinces and cities, with

over 63 people died of more 125 patients was infected by this virus
up to date. Clade 1, 2 and 7 strains belong to Z, V and G genotypes
of the A/H5N1 virus, are widespeared circulated virus groups, cause
A/H5N1 oubreaks in poultry specieses and infected to human, in
Vietnam and many countries in the world from 2003 up to date.
Recently, clade 1.1 and 2.3.2.1 virus group is formed in 2009, have
much different various of the H5 antigenic and high virulence, in
comparision virus strains in the past, and complicate the A/H5N1
influenza oubreaks prevention.
The genetically variation of PB2, PB1 and PA polymerase help
A/H5N1 influenza virus to adapt transcription in infected cells,
decides easilier transmition of virus from human to human, and
2
increases virulence in the body of human. At present, this is one of
problems that being interested in supervising and studying A/H5N1
virus, pathogenic in domestic poultry and human. The genetically
data of PB2, PB1 and PA provide a scientific basic to forecast early
molecular epidemic and using the vaccine, to prevent A/H5N1 virus
in human and domestic poultry specieses.
2. The objective of the topic
- Sequencing, comparising genetically variation and homology
rate of nucleotide and amino acid of PB2, PB1 and PA genes of some
A/H5N1 influenza strains, isolated in Vietnam during 2007 to 2011,
with A/H5N1 strains isolated in the world.
- Identifying phylogenetic relationships of these genes of A/H5N1
strains in this study with A/H5N1 strains in Vietnam and the world.
3. The range of the reseach topic
PB2, PB1 and PA polymerase in the genome of 6 strains belong
to 3 clades: 1.1, 2.3.2.1 and 2.3.4.3 of the A/H5N1 influenza virus,
was collected from 6 corresponding material samples, that contained

A/H5N1 virus of the infected poultry in Vietnam from 2007 to 2011.
4. The layout of the dissertation
The dissertation consists of 116 pages, including: Introdution is 2
pages, Chapter 1 – Overview is 30 pages, Chapter 2 – Subjects and
menthod is 20 pages, Chapter 3 – Results of research is 35 pages,
Chapter 4 – Discussion is 26 pages, Conclusion is 2 pages and
Recommendations is 1 page. List of publication is 1 page,
References 13 pages and Appendix is 39 pages. In dissertation: 29
tables, 26 figures. The dissertation has 118 references, including: 14
documents of Vietnamese, 104 documnents of English and 8 web pages.
3
Chapter 1. OVERVIEW
1.1. BIOLOGICAL CHARACTERISTICS OF THE A INFLUENZA VIRUS
1.1.1. The structure of the A influenza virus
1.1.2. The structure of the genome A influenza virus
1.1.3. Structure and function of RNA segments of the genome
the A influenza virus
1.1.4. Structural and fuctional characteristics of PB2, PB1 and PA genes
1.1.5. The polymerase complex of the A influenza virus
1.1.6. Genetically variation characteristic of genes and the
genome A influeza virus
1.2. A INFLUENZA PANDEMIC AND VARIATIONAL CHARACTERISTIC
OF PB2, PB1 AND PA GENES OF THE HUMAN A
INFLUENZA VIRUS
1.2.1. A influenza pandemics in the past
1.2.2. Variational characteristic of PB2, PB1 and PA of the
human A influenza virus
1.3. EPIDEMIOLOGICAL AND BIOLOGICAL CHARACTERISTIC
OF THE A/H5N1 VIRUS
1.3.1. Epidemiological characteristic of the A/H5N1 virus

1.3.2. Biological characteristic of the A/H5N1 virus
1.4. STUDY OF THE VARIATION OF PB2, PB1, PA GENES
RELATED VIRULENCE AND TRANSMISSION OF THE
A/H5N1 INFLUENZA VIRUS IN HUMAN
1.4.1. In the world
1.4.2. In Vietnam
4
Chapter 2. SUBJECTS AND MENTHODS
2.1. SUBJECTS, MATERIALS AND EQUIPMENTS
2.1.1. The subjects and materials of this study
* Subjects of this study: including PB2, PB1 and PA genes of the
genome of 6 A/H5N1 strains belong to 3 clades: 2.3.4.3, 2.3.2.1 and
1.1, that causing avian influenza oubreaks in the poultry and infected
in the human in Vietnam during 2007 to 2011.
* Materials of this study:
+ In this study used 6 material samples collected from ducks and
chickens were died in A/H5N1 avian influenza oubreaks, that occurs
at some location in Vietnam during 2007 – 2011, to receive
nucleotide and amino acid sequences of PB2, PB1 and PA genes of
the genome of these virus strains.
- All of 6 material samples content corresponding A/H5N1
influenza virus strains, based sequencing and definning results of H5
gene, that was studied and publicated by authors: Nguyen Thi Bich
Nga and Le Thanh Hoa (2012). Material samples and A/H5N1 virus
strain in them, is abbreviation sign and full name as the international
nomenclature in this study (Table 2.1).
Table 2.1. List of six A/H5N1 influenza virus strains in this study
Number
SAMPLES /
VIRUS STRAINS

SIGN
INTERNATIONAL NOMENCLATURE
Year
isolated
CLADE
H5
01. DkQT801-2011 A/Duck/VietNam/QT801/2011(H5N1) 2011 2.3.2.1
02. DkQT802-2011 A/Duck/VietNam/QT801/2011(H5N1) 2011 2.3.2.1
03. DkTG926-2009 A/Duck/VietNam/TG926/2009(H5N1) 2009 1.1
04. CkDT382-2008 A/Chicken/VietNam/DT382/2008(H5N1) 2008 1.1
05. DkNA72-2007 A/Duck/VietNam/NA72/2007(H5N1) 2007 2.3.4.3
06. DkNA114-2007 A/Duck/VietNam/NA114/2007(H5N1) 2007 2.3.4.3
Note: Dk: Duck, Ck: Chicken, QT: Quangtri, TG: Tiengiang, DT: Dongthap, NA: Nghean,
Clade H5: classification of the A/H5N1 virus by clade H5 gene.
5
+ PB2, PB1 and PA genes of 6 A/H5N1 strains in this study, were
collected, analyzed and compared genetically variation with
corresponding genes of 25 A/H5N1 virus strains, belong to 4 virus
groups: clade 1, 1.1, 2.3.4.3 and 2.3.2.1, that isolated during 2007 –
2012 period, in Vietnam and some coutries in the world.
+ The sequence of PB2, PB1 and PA genes of referenced virus
strains, was obtained from online database of Genbank, that have
clade H5 and virus genotype was defined and publicated in
documents in the past.
2.1.2. Tools and equipments
2.1.3. Biological reagent kits use in this study
In this study used biological reagent kits for the molecular biological
technique, that were provided by companies: QIAGEN (USA),
Fermentas (USA), Bioneer (South Korea) and Invitrogen (Japan).
2.1.4. Environment use in cloning

2.1.5. Chemicals use in DNA electrophoresis on agarose
2.2. MENTHODS IN THIS STUDY
Protocol studying for PB2, PB1 and PA genes of the A/H5N1
influenza virus showed in Figure 2.1.
2.2.1. Viral RNA extraction
Using the QIAamp Viral RNA Mini Kit (QIAGEN).
2.2.2. Design oligonucleotide prime sequences used in this study
- The nucleotide sequence of primes in this study were obtained
by using the MacVector 8.2 program and were compared with the
“Primer design” program in Genbank
(
- Prime sequences produced by the Laboratory of the Bioneer
company (South Korean.
6
Figure 2.1. Protocol diagram was using in study PB2, PB1 and PA
polymerase genes of the A/H5N1 influenza virus
2.2.3. RT-PCR technique
- The viral RNA was transcripbed ino cDNA by using 468F and
hexamer primes, according to protocol provided by using the
Maxima™ Universal First Strand cDNA Synthesis Kit (Fermentas),
- Using the PCR technique to obtain DNA sequences of PB2, PB1
and PA genes in the genome virus, with viral cDNA and specific
primepairs, was done by using the PCR Master Mix 2X Kit (Fermentas).
2.2.4. Purification DNA products of RT-PCR/PCR
Purification DNA products of RT-PCR/PCR was done by using
the AccuPrep
®
Gel purification Kit (Bioneer).
7
2.2.5. Electrophoresis nucleic acid

2.2.6. Cloning DNA
Purified DNA products of PB2, PB1 and PA genes were cloned
into the vector PCR2.1-TOPO, by using the TA cloning
®
Kit
(Invitrogen).
2.2.7. Sequencing DNA of the gene and genome
The recombinant plasmid DNA is sequenced by using Big Dye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)
2.2.8. Processing, obtaining nucleotide and amino acid sequence
of genes in this study
The nucleotide sequence of PB2, PB1 and PA genes was
processed by using SeqEd v1.03 and MacVector 8.2 (Accelrys Inc.)
programs on the Macintosh computer.
Analysing, comparing of nucleotide and amino acid sequences
was done by the GENEDOC 2.5 program. Analysing of phylogenetic
relationships was done by using the MEGA4.1 program on the
personal computer.
2.3. THE ETHICAL ASPECTS OF THE RESEARCH
8
Chapter 3. RESEARCH RESULTS
3.1. Obtaining nucleotide sequence of PB2, PB1 and PA
polymerase genes of six A/H5N1 strains in this study
The full nucleotide sequence of PB2, PB1 and PA genes were
obtained, after steps: viral RNA extraction, RT-PCR two steps,
sequencing, processing and comparison with nucleotide sequences of
the corresponding genes of the A/H5N1 virus, that stored in online
data of the Genbank.
Analysis results:
- PB2, PB1 and PA polymerase genes of 6 A/H5N1 strains in this

study, contain 2.280, 2.274 and 2.151 nucleotides, respectively. One
after another theses genes encoding corresponding proteins contain:
759, 757 and 716 amino acids, respectively.
- The PB1 gene of 6 A/H5N1 strains in this study, contains the
PB1-F2 open reading frame (including 273 nucleotides, encode 90
amino acids) and the PB1-N40 open reading frame (including 757
nucleotides, encode 718 amino acids).
- The nucleotide sequence of PB2, PB1 and PA genes were
obtained after sequencing, are 97% - 99% homology rate in 96% -
100% comparison, with corresponding gene sequences of the
A/H5N1 strains stored in online data of the Genbank.
3.2. Comparing nucleotide and amino acid of PB2, PB1 and PA
genes of six A/H5N1 strains in this study with A/H5N1
strains in the world
Nucleotide and amino acid sequences of PB2, PB1 and PA genes
of 6 A/H5N1 virus in this study, were comparised with
corresponding sequences of 19 A/H5N1 strains belong to 4 virus
groups: clade 2.3.2.1, 2.3.4.3, 1 và 1.1.
9
3.2.1. Comparing nucleotide and amino acid of the PB2 gene
3.2.1.1. Comparing nucleotide and amino acid
- The nucleotide sequence of the PB2 gene of 6 A/H5N1 strains
in this study, contains 2.280 nucleotides and encode 759 amino
acids, as nucleotide and amino acid amount as compared with this
gene of above 19 virus strains.
- Apart from many separate different positions, there are 110
positions specific difference of nucleotide in PB2 sequence, between
6 strains of studying and 19 strains represented 4 comparative virus
groups. However, have only above 18/110 nucleotide different
positions lead to change amino acid in the PB2 protein sequence.

- Six A/H5N1 strains of studying and A/H5N1 virus strains
isolated from domestic poultry in 4 comparative virus groups, are
PB2 protein sequence conseved amino acid glutamic acid at the 627
th
position (E627) and aspartic acid at the 701
th
position (D701).
- In particular, PB2 nucleotide sequence of 7 A/H5N1 strains
isolated from patients of clade 1 and 2.3.4.3 virus groups, have a
mutation to change nucleotide (A↔C) at the 1897
th
position, leads to
change amino acid at the 627
th
(E627K) position in the PB2 protein,
in comparison with corresponding sequence of virus strains isolated
from poultry.
3.2.1.2. Comparing homology rate (%) of nucleotide and amino acid
- The PB2 gene homology rate (%) of nucleotide and amino acid,
are 92% – 99% and 96% – 100% respectively, between 25 A/H5N1
virus strains were compared in this study (Table 3.1).
- These rate of the PB2 gene between 6 A/H5N1 virus strains of
studying and represented strains in the 4 virus groups, isolated in
China, Thailand, Cambodia and Lao, are 96% – 99% of the
nucleotide and 98% – 100% of the amino acid (Table 3.1).
10
Bảng 3.1. The homology rate (%) of nucleotide and amino acid of the PB2 gene
Numberica
l
order

CLADE 2.3.2.1 CLADE 2.3.4.3 CLADE 1 CLADE 1.1
1 2 3 4 5 6 7 8 9 10 11 12
1
3
14 15 16 17 18 19 20
2
1
2
2
2
3
24 25
CLADE 2.3.2.1
1 99 98 98 98 96 96 96 96 96 96 96 95 96 95 95 95 94 94 94
9
3
9
3
9
3
9
3
92
2 99 98 98 98 96 96 95 95 95 95 95 95 95 95 95 94 94 94 94
9
3
9
3
9
3

9
3
92
3 99 99 98 99 96 97 96 96 96 96 96 96 96 95 95 95 94 94 94
9
3
9
3
9
3
9
3
9
3
4 99 98 99 98 96 96 96 96 96 96 96 95 96 95 95 94 94 94 94
9
3
9
3
9
3
9
3
92
5 99 99 99 99 97 97 96 96 96 96 96 96 96 95 95 95 95 95 95
9
3
94
9
3

9
3
9
3
6 98 98 98 98 98 99 96 96 96 96 96 96 96 95 95 95 95 95 95
9
3
9
3
9
3
9
3
9
3
7 98 98 99 98 99 98 97 97 97 97 97 97 97 95 95 95 95 95 95 94 94
9
3
94
9
3
CLADE 2.3.4.3
8 98 98 98 98 98 98 98 99 99 99 99 98 99 96 96 96 96 96 96 95 95 95 95 95
9 98 98 98 98 98 98 98 100 99 99 99 98 99 96 96 96 96 96 96 95 95 95 95 95
10 98 97 98 98 98 97 98 99 99 99 99 99 99 96 96 96 96 96 96 95 95 95 95 94
11 98 98 98 98 98 98 98 99 99 99 99 98 99 96 96 96 96 96 96 95 95 95 95 94
12 98 97 98 98 98 97 98 99 99 98 99 98 99 96 96 96 96 96 96 95 95 94 95 94
13 98 98 98 98 98 98 98 99 99 99 99 98 98 96 96 96 96 96 96 95 95 94 95 94
14 98 98 98 98 98 98 98 99 99 99 99 99 99 96 96 96 96 96 96 95 95 95 95 94
CLADE 1

15 97 97 98 97 98 97 98 98 98 98 98 98 98 98 99 98 98 98 98 97 97 97 97 97
16 97 97 98 98 98 97 98 98 98 98 98 98 98 98 99 99 98 99 98 97 97 97 97 96
17 97 97 98 97 98 97 98 98 98 98 98 98 98 98 99 99 99 98 98 96 97 96 97 96
18 97 97 98 97 98 97 98 98 98 98 98 98 98 98 99 99 100 98 98 96 97 96 96 96
19 97 97 97 97 97 97 97 98 98 98 98 97 98 98 99 99 99 99 98 96 97 96 97 96
20 97 97 97 97 97 97 97 98 98 98 98 97 98 98 99 99 99 99 98 96 97 96 96 96
CLADE 1.1
21 96 96 96 96 96 96 96 97 97 96 96 96 97 97 98 98 98 98 97 97 98 98 98 98
22 96 96 96 96 96 96 96 97 97 96 96 96 97 97 98 98 98 98 97 97 98 98 98 98
23 96 96 97 97 97 96 97 97 97 97 97 97 97 97 98 98 98 98 98 98 99 99 98 99
24 96 96 96 96 97 96 97 97 97 96 97 96 97 97 98 98 98 98 97 97 98 98 99 98
11
25 96 96 96 96 97 96 97 97 97 96 97 96 97 97 98 98 98 98 97 97 98 98 99 99
Note: Numbers on the diagonal is homology rate (%) of the nucleotide and below the diagonal is homology rate (%) of the amino acid. The numerical order from 1 to 25
is signs of A/H5N1 virus strains were comparised in this study. 1: DkQT802-2011; 2: DkQT801-2011; 3: A/Dk/VN/LBM140/2012; 4: A/MDk/VN/LBM113/2012;
5: A/Hubei/1/2010; 6: A/GCG/QH/1/2009; 7: A/BHG/MN/X53/2009; 8: DkNA72-2007; 9: DkNA114-2007; 10: A/VN/UT31203A/2007;
11: A/VN/UT31244II/2007; 12: A/VN/UT31394II/2008; 13: A/VN/UT31413II/2008; 14: A/MDk/VN/56/2007; 15: A/Ck/VN/NCVD10/2007;
16: A/VN/UT3028/2003; 17: A/VN/1203/2004; 18: A/VN/UT3040/2004; 19: A/TH/2(SP-3)/2005; 20: A/TH/676/2005; 21: CkDT382-2008; 22: DkTG926-2009;
23: A/MDk/VN/OIE/559/2011; 24: A/KH/U0417030/2010; 25: A/KH/V0417301/2011. The number of 6 A/H5N1 strain of studying was bold .
12
3.2.2. Comparing nucleotide and amino acid of the PB1 gene
3.2.2.1. Comparing nucleotide and amino acid
- The nucleotide sequence of the PB1 gene of 6 A/H5N1 strains
in this study, contains 2.274 nucleotides and encode 757 amino
acids, as nucleotide and amino acid amount as compared with this
gene of 19 strains represented 4 comparative virus groups.
- Apart from many separate different positions, there are 98
positions specific difference of nucleotide in PB1 sequence, between
6 strains of studying and 19 strains represented of 4 comparative
virus groups. However, have only above 6/110 nucleotides different

positions lead to change amino acid in the PB1 protein sequence.
3.2.2.2. Comparing homology rate (%) of nucleotide and amino acid
- The PB1 gene homology rate (%) of nucleotide and amino acid,
are 94% – 99% and 98% – 100% respectively, between 25 A/H5N1
virus strains were compared in this study.
- These rate of the PB1 gene between 6 A/H5N1 virus strains of
studying and represented strains in the 4 virus groups, isolated in
China, Thailand, Cambodia and Lao, are 96% – 99% of the
nucleotide and 98% – 100% of the amino acid.
3.2.2.2. Comparing nucleotide and amino acid of the PB1-F2 gene
- The nucleotide sequence of the PB1-F2 gene of 6 A/H5N1
strains in this study, contains 273 nucleotides as nucleotide and
amino acid amount as compared with this gene of 19 strains
represented 4 comparative virus groups.
- Apart from many separate different positions, there are 12
positions specific difference of nucleotide in PB1-F2 sequence,
between 6 strains of studying and 19 strains represented 4
comparative virus groups (Figure 3.1).
13
Figure 3.1. Comparing amino acid sequences of PB1-F2 protein 25 A/H5N1 influenza virus strain
Note: The sign of 6 A/H5N1 virus strains in this study is bold on grey background; Letters in the target sequence are the sing of the international
name’s amino acid; The codon “STOP” position appears in the PB1-F2 protein in circle; The changed amino acid N66S position in the PB1-F2
protein sequence in vertical frame.
14
- The PB1-F2 protein sequence of the DkQT801-2011 strain
(clade 2.3.2.1), there is a mutation to form “STOP” codon at the 10
th
position, and contains only 9 amino acids. Likewise, clade 2.3.4.3
A/VN/UT31244II/2007 and clade 1.1 A/MDk/VN/OIE/559/2011
strains, contain 79 and 25 amino acids, respectively (Figure 3.1).

- Specially, has a changing amino acid arginin (N) to serin (S) at
the 66 position (N66S) in the PB1-F2 protein, of the clade 2.3.2.1
DkQT802-2011 strain and 2 virus strains: CkDT382-2008 and
DkTG926-2009 (clade 1.1) in this study (Figure 3.1).
- The PB1-F2 protein sequence of 2 virus strains DkNA72-2007
and DkNA114-2007 (clade 2.3.4.3), contains complete 90 amino
acids and conserves arginin at the 66
th
position (N66) (Figure 3.1).
3.2.2.2. Determining of the PB1-N40 open reading frame
Six A/H5N1 virus strain of studying and 19 strains represented 4
comparative virus groups, contain the PB1-N40 open reading frame
intergrated in the PB1 open reading frame.
The PB1-N40 open reading frame was limited from ATG-
initiation codon at 118
th
– 120
th
nucleotide positions to TAG, is a
“STOP” codon, at the end of the PB1 open reading frame. So the
PB1-N40 sequence contains 2.157 nucleotides, encoding PB1-N40
protein contains 718 amino acids, lacking 39 amino acids at the N-
terminal in comparison with PB1 protein.
3.2.2. Comparing nucleotide and amino acid of the PA gene
3.2.2.1. Comparing nucleotide and amino acid
- The nucleotide sequence of the PA gene of 6 A/H5N1 strains in
this study, contains 2.251 nucleotides and encode 716 amino acids,
as nucleotide and amino acid amount as compared with this gene of
19 strains represented 4 comparative virus groups.
15

- Apart from many separate different positions, there are 166
positions specific difference of nucleotide in PA sequence, between 6
strains of studying and 19 strains represented 4 comparative virus
groups. However, have only above 21/166 nucleotide different
positions lead to change amino acid in the PA protein sequence.
3.2.2.2. Comparing homology rate (%) of nucleotide and amino acid
- The PA gene homology rate (%) of nucleotide and amino acid,
are 90% – 99% and 95% – 100% respectively, between 25 A/H5N1
virus strains were compared in this study.
- These rate of the PA gene between 6 A/H5N1 virus strains of
studying and represented strains in the 4 virus groups, isolated in
China, Thailand, Cambodia and Lao, are 93% – 99% of the
nucleotide and 96% – 100% of the amino acid.
3.3. Phylogenetic construction and determining relationships
of PB2, PB1 and PA of 6 A/H5N1 strains in this study
The sequence of PB2, PB1 and PA genes of 6 A/H5N1 influenza
virus strains in this study, were determined phylogenetic
relationships among 50 A/H5N1 represented virus strains, isolated
from many difference host specieses since 1996 up to date.
3.3.1. Analysing phylogenetic relationships of the PB2 gene
- The PB2 gene of 6 A/H5N1 influenza virus strains in this study
and A/H5N1 virus strains isolated from 2003 to 2012, were formed a
evolution branch, that is closed relationship with the clade 0 A/H5N1
group belong to GD influenza virus genotype, including the prior
A/H5N1 virus - A/Goose/Guangdong/1/1996 strain (Figure 3.2).
16
Figure 3.2. Phylogenetic tree between 56 A/H5N1 influenza virus
strains established based on nucleotide sequences of the PB2 gene
Note: The phylogenetic tree established by MEGA4.1 program, Neighbour – Joining menthod
with 1000 bootstrap. Six A/H5N1 influenza virus strains in this study is bold and (◄) sign;

Dk:Duck, BHG: Bar headed - goose: VN: Vietnam, CN: China, Gd: Guangdong, Gx: Guangxi;
TH: Thailand, LA: Lao, ID: Indonesia, HK: HongKong. Z, G, V and GD: genotype of the
A/H5N1 virus; The virus strain represented genotype is square framed.
17
- The PB2 gene of 4 A/H5N1 strains: DkQT801-2011, DkQT802-
2011, DkNA72-2007 and DkNA114-2007, formed a evolution
branch with strains of clade 2.3.2.1 and 2.3.4 virus groups, isolated
in Vietnam, China and Mongolia from 2005 to 2012. This branch
contains PB2 gene of the A/Guangxi/1/2005 (clade 2.3.4) belong to
V genotype of the A/H5N1 influenza virus, and separated to 2
evolution subbranchs (Figure 3.2). In which: PB2 gene of 2 strains:
DkQT801-2011 và DkQT802-2011, with clade 2.3.2.1 virus group
formed a futher evolution subbranch, in comparison with evolution
subbranch contains the PB2 gene of 2 strains: DkNA72-2007,
DkNA114-2007 and clade 2.3.4(2.3.4.3) virus group.
- Similarly, the PB2 gene of 2 virus strains: CkDT382-2008 and
DkTG926-2009, formed a 1 evolution branch with strains of clade 1
and 1.1 virus groups, isolated in Vietnam, Thailand, China and
Cambodia from 2003 - 2011 (Figure 3.2). This branch contains PB2
gene of strains: A/HK/212/2003 (clade 1) belong to Z+ genotype and
A/KH/R405050/2007 (clade 1.1) belong to Z genotype.
3.3.2. Analysing phylogenetic relationships of the PB1 gene
- The same PB2 gene, the PB1 gene of 6 A/H5N1 influenza virus
strains in this study and A/H5N1 virus strains isolated from 2003 to
2012, were formed a evolution branch, that is close relationship with
the clade 0 A/H5N1 virus group belong to GD genotype (Figure 3.3).
- The PB1 gene of 4 A/H5N1 strains: DkQT801-2011, DkQT802-
2011, DkNA72-2007 and DkNA114-2007, formed a evolution
branch with strains of clade 2.3.2.1 and 2.3.4 virus groups, isolated
in Vietnam, China and Mongolia from 2005 to 2012. This branch

contains PB1 gene of the A/Guangxi/1/2005 (clade 2.3.4) belong to
V genotype of the A/H5N1 influenza virus (Figure 3.3).
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Figure 3.3. Phylogenetic tree between 56 A/H5N1 influenza virus
strains established based on nucleotide sequences of the PB1 gene
Note: The phylogenetic tree established by MEGA4.1 program, Neighbour – Joining menthod
with 1000 bootstrap. Six A/H5N1 influenza virus strains in this study is bold and (◄) sign;
Dk:Duck, BHG: Bar headed - goose: VN: Vietnam, CN: China, Gd: Guangdong, Gx: Guangxi;
TH: Thailand, LA: Lao, ID: Indonesia, HK: HongKong. Z, G, V and GD: genotype of the
A/H5N1 virus; The virus strain represented genotype is square framed.
19
- Similarly, the PB1 gene of 2 virus strains: CkDT382-2008 and
DkTG926-2009, formed a 1 evolution branch with strains of clade 1
and 1.1 virus groups, isolated in Vietnam, Thailand, China and
Cambodia from 2003 - 2011 (Figure 3.3). This branch contains PB1
gene of strains: A/HK/212/2003 (clade 1) belong to Z+ genotype and
A/KH/R405050/2007 (clade 1.1) belong to Z genotype (Figure 3.3).
3.3.2. Analysing phylogenetic relationships of the PA gene
- The same PB2 and PB1 genes, the PA gene of 6 A/H5N1
influenza virus strains in this study and A/H5N1 virus strains isolated
from 2003 to 2012, were formed a evolution branch, that is close
relationship with the clade 0 A/H5N1 virus group belong to GD
genotype (Figure 3.4).
- The PA gene of 4 A/H5N1 strains: DkQT801-2011, DkQT802-
2011, DkNA72-2007 and DkNA114-2007, formed a evolution
branch with strains of clade 2.3.2.1 and 2.3.4 virus groups, isolated
in Vietnam, China and Mongolia from 2005 to 2012. This branch
contains PA gene of the A/Guangxi/1/2005 (clade 2.3.4) belong to V
genotype of the A/H5N1 influenza virus, and separated to 2
evolution subbranchs (Figure 3.4). In which: PA gene of 2 strains:

DkQT801-2011 và DkQT802-2011, with clade 2.3.2.1 virus group
formed a futher evolution subbranch, in comparison with evolution
subbranch contains the PA gene of 2 strains: DkNA72-2007,
DkNA114-2007 and clade 2.3.4(2.3.4.3) virus group.
- The PA gene of 2 virus strains: CkDT382-2008 and DkTG926-
2009, formed a 1 evolution branch with strains of clade 1 and 1.1
virus groups, isolated in Vietnam, Thailand, China and Cambodia
from 2003 - 2011 (Figure 3.4). This branch contains PA gene of
strains: A/HK/212/2003 (clade 1) belong to Z+ genotype and
A/KH/R405050/2007 (clade 1.1) belong to Z genotype (Figure 3.4).
20
Figure 3.4. Phylogenetic tree between 56 A/H5N1 influenza virus
strains established based on nucleotide sequences of the PA gene
Note: The phylogenetic tree established by MEGA4.1 program, Neighbour – Joining menthod
with 1000 bootstrap. Six A/H5N1 influenza virus strains in this study is bold and (◄) sign;
Dk:Duck, BHG: Bar headed - goose: VN: Vietnam, CN: China, Gd: Guangdong, Gx: Guangxi;
TH: Thailand, LA: Lao, ID: Indonesia, HK: HongKong. Z, G, V and GD: genotype of the
A/H5N1 virus; The virus strain represented genotype is square framed.
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Chapter 4. DISCUSSION
4.1. Results of obtaining nucleotide sequence of PB2, PB1 and
PA polymerase genes of six A/H5N1 strains in this study
Six A/H5N1 strains in this study, including: DkNA72-2007,
DkNA114-2007, CkDT382-2008, DkTG926-2009, DkQT801-2011
and DkQT802-2011, there are PB2, PB1 and PA genes obtained after
sequencing contain 2.280, 2.274 and 2.151 nucleotides respectively,
with ATG initiation codon and TAG stop codon. These is nucleotide
amount of PB2, PB1 and PA genes of the A/H5N1 virus genome
isolated, reseached and published since 1996 to present. This result
showed that there aren’t mutations to change the nucleotide amount

of the above genes of 6 A/H5N1 strains in this study, compared with
corresponding genes of the A/H5N1 virus as from was formed the
virus to present.
4.2. Results of comparing nucleotide and amino acid
sequences of PB2, PB1 and PA genes of six A/H5N1
strains in this study with virus strains of the world
- The PB2 sequence of 6 A/H5N1 virus strains, conserved amino
acid at E627 and D701 positions. This result shows that, PB2 gene of
6 A/H5N1 virus strains in this study there isn’t a mutation to help the
virus adapt to transcription easily in the human infected cells.
- The PB1-F2 protein of the DkQT801-2011 strain, has only 9 amino
acid. Whereas, this protein of DkQT802-2011, CkDT382-2008 and
DkTG926-2009 strains, have a mutation to change amino acid N66S. So
that, PB1-F2 protein of these strains there was mutations related to
change the virulent of the virus, leads to being more and more
complicated and difficult to predict of epidemiology and pathology
problems of the A/H5N1 influenza virus.
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