Tài liệu 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. 1 ABREVIATIONS IN THE DISSERTATION bp Da DNA dNTP ddNTP FAO HA kb M MEGA NA NEP NP NS OIE PA PB1 PB2 RT-PCR RNA RNP (-) ssRNA WHO base pair dalton Deoxyribonucleic acid Deoxy Nucleotide Triphosphate dideoxy Nucleotide Triphosphate Food and Agricultural Organization Hemagglutinin Kilo base Matrix protein Molecular Evolutionary Genetics Analysis Neuraminidase Nuclear Export Protein Nucleoprotein Non-structural protein Office International des Epizooties Polymerase acidic protein Polymerase basic protein 1 Polymerase basic protein 2 Revertranscription Polymerase Chain Reaction Ribonucleic acid Ribonucleoprotein Negative single-strand Ribonucleic Acid World Health Organization 2 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 3 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. 4 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 5 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. 6 + 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 (http://www.ncbi.nlm.nih.gov/Primer.cgi). - Prime sequences produced by the Laboratory of the Bioneer company (South Korean. 7 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). 8 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 9 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. 10 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 701th 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 627th (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). 11 Bảng 3.1. The homology rate (%) of nucleotide and amino acid of the PB2 gene CLADE 2.3.2.1 Numberica l order 1 CLADE 2.3.2.1 1 2 99 3 98 4 98 5 98 CLADE 2.3.4.3 6 96 7 96 96 96 97 96 97 99 CLADE 1 CLADE 1.1 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 96 96 96 96 95 96 95 95 95 94 94 94 93 93 93 93 92 25 CLADE 1.1 CLADE 1 CLADE 2.3.4.3 2 99 98 98 98 96 95 95 95 95 95 95 95 95 95 94 94 94 94 93 93 93 93 92 3 99 99 98 99 96 96 96 96 96 96 96 96 95 95 95 94 94 94 93 93 93 93 93 4 99 98 99 98 96 96 96 96 96 96 95 96 95 95 94 94 94 94 93 93 93 93 92 5 99 99 99 99 97 96 96 96 96 96 96 96 95 95 95 95 95 95 93 94 93 93 93 6 98 98 98 98 98 96 96 96 96 96 96 96 95 95 95 95 95 95 93 93 93 93 93 7 98 98 99 98 99 98 97 97 97 97 97 97 97 95 95 95 95 95 95 94 94 93 94 93 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 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 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 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; 12 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 . 13 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). 14 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. 15 - 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 66th 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 ATGinitiation codon at 118th – 120th 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 Nterminal 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. 16 - 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). 17 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.