1 INTRODUCTION The imperative research of the subject Highly pathogenic Avian Influenza (HPAI) caused by the A/H5N1 influenza virus is an acute infectious disease of poultry The Influenza virus A/H5N1 belongs to type A of the Orthomyxoviridae family, possessing two proteins, ie., hemagglutinin (HA) and neuraminidase (NA) on the surface of the virion capsid, involving in the immune response There are 16 HA subtypes (H1 to H16) and NA subtypes (N1 to N9) The HA antigenic polypepide is encoded by segment of the genome of influenza A virus, which is also an attachment polypeptide to a specific receptor on the membrane surface of the infected cells HA has the ability to genetically vary to generate mutations in the gene and subsequent amino acid changes the polypeptide at the "antigenic site 2” (position 152-157) and the protease enzyme cutting site between HA1 and HA2 or going to reassortment to perform a new/modified isolates (newly performed reassortants) The NA antigenic polypepide is encoded by segment This invloves in enzymatic process by cutting glycoside links at sialic acid molecules (Nacetylneuramic acid) at the receptor to release viral progeny during virus infection In Vietnam, avian influenza outbreaks have appeared since late 2003, early 2004 and spread over many localities in the whole country Major proportions of poultry and aquatic birds have died or been culled or destroyed nationwide during the early years of the endemic causing enormous affection to the nation economy Investigations on variations of the genetic materials in the genome of the virus, therefore, are essential, in order to gain knowledge about the molecular changes of influenza A/H5N1 virus, especially the characteristics of the HA antigenic genes (H5) and NA (N1) in those strains which have been isolated in Vietnam during 2004 and 2011 The resulting achievements can give rise to assess the viral evolution associated with the antigenic changes and to make predictions in every aspects of the molecular epidemiology, the studies on genetic orientation, the vaccine developent and the use of avian influenza vaccine for appropriate effectiveness Derived from these requirements, we proceeded to implement the theme entitled: “Molecular characterization of the H5 and N1 genes of the A/H5N1 avian influenza virus isolated in Vietnam for new generation vaccine development The objects of the theme Sequencing the full H5 and N1 genes of influenza A/H5N1 isolates collected in different locations in Vietnam during 2004 to 2011 Cloning H5 gene and N1 gene into the vectors for maintenance of the genetic materials for new generation vaccine development Analysis of molecular characteristics of the H5 and N1 antigen genes, identifying phylogenetic relationships with strains of influenza A/H5N1 in Vietnam and the world Novel contributions of the dissertation This dissertation describes the sequencing and comparative analysis of the complete hemagglutinin and neuraminidase of each strain of the A/H5N1 viruses isolated from provinces in Vietnam during 2004 – 2011 By using molecular approaches, incluidng RT-PCR, cloning, and sequencing, the complete nucleotide and amino acid sequence for hemagglutinin and neuraminidase, respectively, for the isolated A/H5N1 viruses was obtained and analyzed Phylogenetic results revealed that infection of strains from multiple sublineages and clades occured in each period of endemics Since the first detection of the A/H5N1 in Vietnam, among our studied strains, there were clades determined, ie., clade 1, clade 1.1, clade 2.3.2.1 and clade 2.3.4.3, of which clade 1.1 (since 2008) and clade 2.3.2.1 (since 2011) have recently emerged in Vietnam Research objectives and outlines The sampling strategy was to collect mucosal (exudate), tracheal, nasopharyngeal materials of the infected poultry that contained virulent avian influenza A/H5N1 viruses and inactivated by high temperature and stored at -20oC until use The range of the research topics is: i) to decode the H5 and N1 genes; ii) to analyze nucleotide and component amino acid sequence of the H5 and N1; iii) to establish the phylogenetic relationship of the strains obtained with the strains of Vietnam and the world registered in GenBank; iv) to detect new clades recently emerged in Vietnam Arrangement of the dissertation The thesis consists of 99 pages, including the page- Introduction; 35 pageLiterature Overview; 11 page- Materials and Methods; 48 page- Results and Discussion; page- Conclusions and Recommendations, and a List of publication; 12 page- References; 39-page Appendix CHAPTER LITERATURE OVERVIEW 1.1 Outline of the influenza virus 1.1.1 Influenza viruses and their classification 1.1.2 Evolutionary history forming the different HPAI sublineages of the avian A/H5N1 viruses 1.1.3 The formation of the genotypes of the influenza A/H5N1 virus in the world and in Vietnam 1.1.4 Genetic variation in hemagglutinin (HA) forming clades of the avian A/H5N1 viruses 1.2 Structural features of the influenza A virus 1.2.1 Common structural characteristics 1.2.2 Genome structure of the influenza A virus 1.3 Structure and function of the hemegglutinin and the neuraminidase 1.3.1 HA (hemagglutinin) protein 1.3.2 NA (neuraminidase) protein 1.4 The virulence determining factors 1.5 The ways of antigenic variation 1.5.1 Antigenic drift 1.5.2 Antigenic shrift 1.5.3 Glycosylation 1.6 Introduction of the avian influenza 1.6.1 History of the avian influenza 1.6.2 Overall situation of the A/H5N1 avian influenza in the world 1.6.3 Overall situation of the A/H5N1 avian influenza in Vietnam 1.6.4 Characteristics of the A/H5N1 avian influenza virus 1.6.5 Multi-host adaptivity of the A/H5N1 avian influenza virus 1.6.6 Mechanism of infection and replication of the influenza A virus in a cell 1.6.7 Transmission of the avian influenza A/H5N1 virus 1.6.8 Resistance of the avian influenza A/H5N1 virus 1.7 Vaccine for poultry to protect avian influenza 1.8 Overall studies on the influenza virus A/H5N1 in Vietnam CHAPTER SUBJECTS, CONTENT AND METHODOLOGY 2.1 The subjects H5 and N1 genes amplified from 14 strains of the influenza A/H5N1 virus causing bird flu in Vietnam collected from a number of localities during the years from 2004 to 2011 The specimens of 14 strains collected in Hanoi, Nghe An, Quang Tri, Khanh Hoa, Vinh Long, Dong Thap, Soc Trang, Tra Vinh, Kien Giang, An Giang 2.2 Contents Sequencing the full H5 and N1 genes of influenza A/H5N1 and cloning into the vectors; Analysis of characteristics of the H5 and N1 antigen genes, phylogenetic relationships in Vietnam and the world 2.3 Materials They were suspensions of samples of which RNA was extracted 2.4 Chemicals/Reagents Chemicals/Reagents used in this study were provided by Qiagen, Invitrogen, Fermentas, Bioneer 2.5 Methods for studying on molecular characterization of H5 and N1 genes of influenza A/H5N1 virus 2.5.1 Genomic RNA extraction: Genomic RNA was extracted using QIAamp Viral Mini Kit (QIAGEN), according to the instruction from the manufacterer 2.5.2 RT-PCR application: Full lenght of H5 and N1 genes were amplified using QIAGEN OneStep RT-PCR kit (QIAGEN) 2.5.3 Electrophoresis and purification of RT-PCR products: RT-PCR products were visualized on 1% agarose; and purified using QIAquick PCR purification kit (QIAGEN) 2.6 TA-cloning of the RT-PCR products and extraction of recombinnat plasmid DNA Purified PCR products were cloned into the vector pCR2.1; extraction of recombinant plasmid DNA, sequenced on an ABI-3100 Avant Genetic Analyzer 2.7 Sequencing and data processing The chromatograms and sequences were processed by SeqEdv1.03, AsemblyLIGNv1.9c and MacVector8.2 (Accelrys Inc.) using a Macintosh computer Analysis and comparison of nucleotide and amino acid composition and analysis of phylogenetic relationships were done by using GENEDOC2.5 programs and MEGA4.0 on a PC computer CHAPTER RESULTS AND DISCUSSION 3.1 Obtaining hemagglutinin sequences and cloning 3.1.1 Hemagglutinin subtype products and their sequencing Using RT-PCR (Qiagen) with primerpair H5F-H5R, complete H5 sequences were obtained from all 14 strains in this study: H5F: 5’-TCTGTCAAAATGGAGA AAATAGTG-3’ and H5R: 5’-TTAAATGCAAATTCTGCATTG-3’ As a result, RT-PCR products were of about 1707 nucleotides in length coding for 568 amino acid for the strains of Guangdong sublineage and 1704 nucleotides encoding 567 amino acids for strains of the Fujian sublineage (Figure 3.8) Results of 14 strains analyzed in our study showed that there are 41 positions where major amino acid differences between strains were inspected The differences were mainly in the HA1, ie., 32 positions (10, 11, 18, 61, 69, 96, 102, 104 110, 136, 140, 149, 156, 170, 171, 172, 178, 179, 190, 200, 205, 216, 228, 235, 242, 243, 256, 285, 293, 298, 326, 341) and in the HA2 there were only positions (345,375, 462, 473, 491, 524, 529, 544 , 549) All strains of the Guangdong sublineage during 2004-2005 period, have the common motif (RRRKK) at the HA1-HA2 connecting region; however, the motif has changed to (GRRKK) in the strains collected in the period after 2006 while the Fujian strains have the motif (RRRK) Amino acid composition of the H5 protein DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : * 20 I * 40 II * 60 * MEKIVLLFASISLVKSDHICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAG T L.I .Q D R R .IV Q D R R .L.IV R Q D .R .IV Q .T D R R E IV Q D R R .IV R.Q D R R .L.I .Q D .R .L.I .Q E .R .IV Q D .R .L.I .Q.Y R .D .R .L.I .Q D .R .IV Q D .R .IV Q D .R .L.I .Q D .R .L.I .Q D .R .L.IV Q D .R .IV Q D .R .IV Q D .R .IV Q D.G R R IV Q D .R FL.IV Q D .R .IV Q D .R .IV Q D .R .L.TV Q R .R .IV Q G A.D .R L IV Q R .D .R .IV Q D .R .IV Q .K D .R T L.TV Q R .R R L.IV Q R : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 75 DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 80 * 100 * 120 * 140 * WLLGNPMCDEFTNVPEWSYIVEKANPANGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASLGVSAA P G .I D .N I V.D V .S PS .I V.D V .S PS .I D S S S S .I V.D V .S PS .I D V .S S .I V.D V .S PS .I D L R .S S S .I D .N S S .I V.D D .S S S .I D .S S SV I D .S S S .I V.D D .S S S .I V.D V .S PS .I D .S S S .I D D .S S S .I D .S S .I V.D V .S PS .I V.D V .S PS .I V.D D .S S S .I V.D D .S S .I D .S S S .I V V.D V .S S S .I V V.D V .S S S .I S D .S N.D S S .I V.D D .S S S .I V.D D .S S S .I V.D D .S S S .I V.D D .S S S .I S D .S N.D S S .I S D D T PS N.D S S : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 150 DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 160 *III 180 IV * 200 * 220 CPYQGSSSFFRNVVWLIKKDNAYPTIKKDYNNTNQEDLLILWGIHHPNDEAEQTRLYQNPTTYISIGTSTLNQRL DNA .K DNA G V .K NST RS D VM A K V .L.K NST RS VM A IK V TP NNT RS V S AT IK V.V K D .NST RS D VM A K V K NST RS D VM A AK I V K NST RS D VM A K V VP D .NNT RS .S A K V TP NNT RS .S A K V K NST RS V .A K V VP I.NNT RS .S A K V D .VP NNT RS .S A K V D .K NST RS V .A K V K NST RS VM A K V VP NNT RS .S A K V VP NNT RS .S A K V MP NNT RS .S A K V K NST RS VI A K V K NST RS VM A K K NST RS V W A K V K NST RS VM A K V TP NNT RS .S A K V K NST RS VI A IK V K NST RS VI AT IK V .L.R NST RS V .A K V K NST RS V .A IK V K NST RS V .A K V K NST RS V .A K V K NST RS V .A K V .L.R NST RS V .A K V .H.R NST RS V .A K V : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 225 DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF : : : : : : : : : : : : : : : : : : : : : : : * 240 * 260 * 280 * 300 VPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPIGA T.R V ME N L .K M T.R V ME .N N L .K M V M S N A S.K T.R V ME F N L .K M T.R V ME N L .K M T.R V ME N L .K M V M N M .K .V M N A K R V ME N L .K M V M N A K V V M N A K R V ME N L .K M T.R V ME N L .K V V M N A K .V M N A K .V M N A G K T.R V ME N L .K M T.R V ME N L .K M R V ME N L .K M R V ME N L .K M : : : : : : : : : : : : : : : : : : : : : : : 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : V M N A.I K R V ME N L .K M R V ME N GL .K M E P.V ME N L .K M R V ME N L .K M R V ME N L .K M R V ME N L .K M R V ME N L .K M E P.V ME N L .K M E P.V ME N A L .K M DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : V * 320 * 340 * 360 * INSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRERRRK-RGLFGAIAGFIEGGWQGMVDGWYGYHHSND R P RRRK- RRRK- E R GRRRK E TR GRRRK E S L RRRK- E .L D R GRRRK E L R GRRRK E R GRRRK E .L RRRK- F E .L RRRK- F E R RRRKK E .L RRRK- S F E .L RRRK- F E R RRRKK E R GRRRK E .L RRRK- F E .L RRRK- F E .L RRRK- E R GRRRK E R GRRKK E H R RRRKK E L R RRRKK E .L RRRK- E R K.RRRKK E R K.RRRKK E R .A RRRKK .R RRRKK E R RRRKK E R RRRKK E R RRRKK E R .T RRRKK E R .T RRRKK N .E DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 : : : : : : : : : : : : : HA1 : : : : : : : : : : 300 300 300 300 300 300 300 300 300 300 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 374 374 374 375 375 374 375 375 375 374 374 375 374 374 375 375 374 374 374 375 375 375 375 374 375 375 375 375 375 375 375 375 375 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 449 449 449 450 450 449 450 450 450 449 449 450 449 449 450 450 449 449 449 450 450 450 450 449 450 450 450 450 450 450 450 450 450 : : : : : : : : : : : : : 524 524 524 525 525 524 525 525 525 524 524 525 524 HA2 380 * 400 * 420 * 440 * QGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN .R .S N H .PPNQK LN.F.I FT IT N Q 460 * 480 * 500 VI * 520 ERTLDFHDSNVRNLYDKVRLQLKDNAKELGNGCFEFYHKCNNECMESVRNGTYDYPQYSEEARLKREEISGVKME D L .K R .D L .K D L .K R .D L G K R .D L .K R .D.K L .K R .D I L .K R .D L .K R .DD K L .K R .D L .K A R .D L 8 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : .K R .D L .K R .D L .K R .D L .K R .D L .K R .D L .K R .DD .L .K R .D L .K R .D L .K R .DD .L .K R .D L .K R .D L .K R .D L .K R .D L .K R .D .K V N L .K R .D L .K R .D L .K R .D L .K R .D L .K R .D .K .N L .KH .R .D .K .N L DkQT801-20 DkQT802-20 A-Hubei-1( CkKH-2010 Dk0970(09) Dk-VN-TMU0 CkDT382(08 CkKG88(08) CkTV98(08) Ck-VN-TMU0 A-VN-UT313 CkTL-CU-35 DkNA72-07 DkNA114-07 DkMB2-07 DkBL(07)GU DkVN50(07) A-VN(07)EU A-FJ1(07)F DkCM(06)EU MdBentre34 A-TL(06)GQ A-VN(05)EF A-FJ1(05)F DkAG-05 CkVL-05(17 DkVN1(05)D CkHD1-04-E MdGL-04 A-VN-1194A-VN-1203A-HK156(97 GsGD1(96)A : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : * 540 * 560 VII SIGIYQILSIYSTVASSLVLAIMMAGLSLWMCSNGSLQCRICI .V A V V A V T V V A LV V.V A V V A I T A V T A V A V T A V T A V A V A V T A V T A V T A V A V A V A V A V T A V A I A I M.T A V A V A V H .A V A V M.T A V M.T A V - : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 524 525 525 524 524 524 525 525 525 525 524 525 525 525 525 525 525 525 525 525 567 567 567 568 568 567 568 568 568 567 567 568 567 567 568 568 567 567 567 568 568 568 568 567 568 568 568 568 568 568 568 568 568 Figure 3.1 Alignment of amino acid sequences of H5 polypeptide for 33 strains (including 14 strains isolated in Vietnam from 2004 to 2011 in this study) with the sequences of the strains in Genbank Note: Dot (.) denotes the sequence identical to the corresponding amino acid sequence of the first H5 (DkQT801-2011) The difference in amino acid represented by the symbols of our alphabet The amino acids involved in the protease cleavage site is framed The arrow indicates the direction of the HA1-HA2 assignment The position of the glycosylation sites are underlined and numbered by Roman I through VII Regions responsible for receptor-binding is in bold 3.1.2 Analysis of glycosylation position, protease cleavage, adhesion receptorbinding and epitopes of the H5 polypeptide sequences List of key amino acid positions determining important characteristics of H5 polypeptide, including the glycosylation protease cleavage, adhesion receptorbinding and epitopes is presented in Table 3.1 Analysis was done for the entire 568 amino acids of H5 polypeptide of the Guangdong sublineage (clade 1), 567 amino acid of the Fujian sublineage (clade 2.3.4) and 32 species of clade 2.3.2 (including 26 strains of Vietnam, strains of China, strains from Thailand Included in the analysis was also 14 virulent strains isolated by us during period 2004-2011 The results showed that there were glycosylation sites (denoted I-VII), in which five were located in HA1 and two in HA2 regions (Figure 3.1) There were identical amino acids constructing glycosylation sites: site I (NNS), II (NVT), IV (NNT), V (NSS), VI (NGT), VII (NGS) (Figure 3.1), but only in site III is difference found (170-172) That is in the Guangdong strains it was NST, and the Fujian strain it is the NNT Particularly, in two strains of clade 2.3.2 (A/Dk/VN/QT801/2011(H5N1) and A/Dk/VN/QT802/2011(H5N1), glycosylation at this site is absent, which is replaced by: D (aspartic acid), N (Asparagine) and A (Alanine), as found in all the strains of clade 2.3.4 and 2.3.2 (Table 3.1) Table 3.1 The characteristic amino acid positions for H5 polypeptide Clade Strains Countries/ Territories Protease cleavage site 338-345/346 A/Duck/Vietnam/QT801/2011* A/Duck/Vietnam/QT802/2011* A/Hubei/1/2010 A/duck/Vietnam/TMU021/2009 A/chicken/Vietnam/TMU004/2008 A/Vietnam/UT31394II/2008 A/Duck/Vietnam/NA72/ 2007* A/Duck/Vietnam/NA114/2007* A/duck/Vietnam/50/2007 A/Vietnam/HN31242/2007 A/Fujian/1/2007 A/Fujian/1/2005 A/Chicken/Vietnam/KH/ 2010* A/Duck/Vietnam/0970/ 2009* A/Duck/Vietnam/DT382/2008* A/Chicken/Vietnam/KG88/2008* A/Ck/Vietnam/TV98/2008 * A/duck/BacLieu/07-09/2007 A/Duck/Vietnam/Ca Mau/498A/2006 A/Muscovy Duck/Vietnam/ BenTre/ 342/2006 A/Chicken/Thailand/CU-354/2008 A/Duck/Vietnam/MB2/ 2007* A/Thailand/NBL1/2006 A/Vietnam/CL2009/2005 A/Duck/Vietnam/AG/2005* A/Chicken/Vietnam/VL/ 2005* A/Chicken/Vietnam/HD1/2004* A/Muscovy Duck/Vietnam/GL/2004* A/Vietnam/1194/2004 A/Vietnam/1203/2004 A/Duck/Vietnam/1/2005 A/HongKong/156/1997 A/Goose/Guangdong/1/1996 Recept orbindin g site 238240 Glycosyl ation position Epitope sites 69 99-102 140-145 152-157 170-172 Vietnam Vietnam China Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam China China Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam 2.3.2.1 QRERRRK-R QSG K ANPA DHEASL PYQGSS DNA 2.3.2.1 QRERRRK-R QSG K ANPA DHEASL PYQGSS DNA 2.3.2.1 QRERRRK-R QSG K ANPA DHEASL PYQGSS DNA 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGTP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGTP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT 2.3.4 LRERRRK-R QSG R ANPA DHEASL PYQGMP NNT 2.3.4 LRERRRK-R QSG R ANPA DHEASS PYQGTP NNT 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST 1.1 QREGRRKKR QSG R ANPA SHEASL PYQGKS NST 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST 1.1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST Thailand Vietnam Thailand Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Vietnam Hongkong China QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QKERRRKKR QSG R VNPV SHEASL PYQGKS NST QKERRRKKR QSG R VNPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ANPV SHEASL PYQGKS NST QRERRRKKR QSG R ASPA NHDASS PYLGRS NST QRERRRKKR QSG R ASPA NHDASS PYLGRS NSA QRERRRKKR QSG R ASPA NHDASS PYHGRS NSA Note: The symbol (*) are the strains isolated and identified in this study 10 - Considering the receptor-binding sequence it indicated that all strains of the many clades circulating in Vietnam, have “QGS” structure and this motif is not changed - Epitopes of the two strains A/Dk/VN/QT801/2011(H5N1) and A/Dk/VN/QT802/2011(H5N1) differ at position 69, between K and R Positions 99-102 of the strains have been circulating in Vietnam from 2004 to present as ANPA, different from strains of classical groups of clade (Gs-GD1-96; AKH156-97 and DkVN1-05 ) have ASPA Epitopes at positions 140-145 in the strains circulating in Vietnam in clade 2.3.2 (and 2.3.2.1), clade 2.3.4 (and 2.3.4.3) and clade (and 1.1), were mainly DHEASL and SHEASL, and major differences were D140S, completely different to the classical strains (NHDASS) (Table 3.1) Epitopes at positions 152-157, showing the amino acid differences seen as PYQGSS in strains emerged in 2011 in North Vietnam (A/Dk/VN/QT801/2011(H5N1) and A/Dk/VN/QT802/2011(H5N1)) or PYQGTP (A-VN -UT31394II-08) or PYQGVP (Ck-VN-TMU004-08) or PYQGKS (A-VN1194-04) and PYHGRS (classic strain Gs-GD1-96) (Table 3.1) Major concern is that the majority of classical strains isolated before 1997 were used to generate masterseed of vaccine, such those including some imported vaccines being used in Vietnam (H5N1 vaccines: the original strain is A-Gs-CNGD1-96(H5N1) and H5N2 vaccine strains are the original a-Turkey-ENG-N2873(H5N2)) Currently, two strains of A-VN-1194-2004(H5N1) and A-VN-12032004(H5N1) were used to generate vaccines as recommended by WHO These strains were used to supply the HA and NA genes, of which the amino acids at protease cleavage sites in H5 were modified by reverse genetics to create vaccines namely NIBRG-14 3.1.3 Phylogenetic construction and relationships among the Vietnamese isolates and the global strains Phylogenetic trees for genetic relationships among strains (Vietnamese and global) are shown in Figure 3.2 (based on nucleotide comparison) and Figure 3.3 (based on comparison of amino acids) Of the 59 strains, there were 44 strains of Vietnam, 10 strains from China, strains from Thailand and a strain of Laos Results shown in Figure 3.2 and Figure 3.3 revealed that: - Two strains solated from ducks in 2011, ie., A/Dk/VN/QT801/2011(H5N1) and A/Dk/VN/QT802/2011(H5N1), were confirmed to belong to clade 2.3.2, which emerged since 2009, previously reported in Vietnam Although the two strains 11 together are similar in terms of the same nucleotide sequence and amino acids, but there are differences in their placement the phylogenetic tree (Figure 3.2 and Figure 3.3) H5 NUCLEOTIDE Ck/VN/DT382(08)JN021302 34 54 58 Ck/VN/TV98(08)JN021300 Ck/VN/KG88(08)JN021301 100 Dk/VN/ST0970(09)JN021303 93 Ck/VN/KH(2010)JN021304 Dk/VN/BL(07)GU052526 100 Dk/VN/CM498A(06)EU124164 63 58 Md/VN/BT342(06)EU124167 66 Dk/VN/ST(05)EU124175 99 A/VN(05)EF456799 Dk/VN/DT680A(05)U124134 79 100 Dk/VN/AG(05)EF051514 Ck/VN/VL(05)EF057808 45 57 Ck/VN/DT171(04)DQ099759 Clade Ck/VN/TG023(04)DQ099758 89 Dk/VN/MB2(07)JN021299 41 83 20 Ck/VN/HD1(04)EF057807 Dk/VN15(03)DQ497670 A/Thailand/NBL1(06)GQ466176 14 100 A/VN/Hanoi30408(05)AB239125 99 Ck/VN8(05)CY016851-clade0 15 Ck/Thailand/CU354(08)CY047457 38 A/VN1203(04)EF541403 A/VN1194(04)EF541402 98 Md/VN/GL(04)EF051515 Ck/Thailand/NIAH108(05)AB450565 43 Dk/HK821(02)AY676033 99 Bh/Goose/Qinghai/67(05)DQ095623 100 Dk/VN/QT801(2011)JN986881 100 Dk/VN/QT802(2011)JN986882 100 A/Hubei/1(2010)CY098758-clade2.3.2.1 Md/VN18159(09)JN055369 61 Dk/VN27373(09)JN055388 100 Ck/VN10(05)CY016867 96 94 Clade 2.3.2 Dk/VN568(05)DQ320839 98 76 Md/VN1455(06)CY029535 Dk/China/E319/2(03)AY518362 Dk/VN/TMU021(09)DB-CY095695 85 36 A/FJ1(05)FJ492882 99 Ck/FJ584(06)DQ992831 A/FJ1(07)FJ492883 42 Gs/GX1898(06)DQ993024 45 Dk/Laos22(06)CY041043 34 Ck/VN/TMU004(08)HN-CY095680 100 47 46 91 Dk/VN/NA72(07)JN021305 Dk/VN/NA114(07)JN021306 Dk/VN50(07)CY029711 43 A/VN/HN31242(07)EU294370 Ck/VN/NCVD/8(03)EF541407-clade5 98 Clade Swine/FJ/F1(01)AY747617-clade5 99 100 100 Clade 2.3.4 A/VN/UT31394II(08)HM114593 54 Dk/VN1(05)DQ366306 A/HK483(97)AF046097-clade0 A/HK156(97)AF046088 Clade Gs/GD1(96)AF148678 100 65 Ck/VN/NCVD/093(08)FJ842480 Ck/VN17(08)FJ842478 Clade Dk/QX/22(01)AY585364-clade3 82 Gs/VN113(01)GU052105 97 100 Clade Gs/VN/324(01)GU052113 0.005 Figure 3.2 Phylogenetic tree between the influenza A/H5N1 strains established based on the nucleotide sequence of H5 using MEGA4.0 program (NJ - Neighbour Joining, testing 1000 bootstraps) Arrows indicate the strains isolated by us used for analysis in this study - Ten strains, comprising of CkKH-2010; Dk0970-09; CkDT382-08; CkKG88-08; CkTV98-08; DkMB2-07; DkAG-05; CkVL-05; CkHD1-04; MdGL04, were grouped in clade with those strains of the Guangdong.sublineage origin - Two strains ie., DkNA72-07 and DkNA114-07 were grouped with strains of clade 2.3.4 of the Fujian sublineage 12 35 77 41 74 H5 AMINO ACID 31 Ck/VN/DT382(08)JN021302 Ck/VN/TV98(08)JN021300 Ck/VN/KG88(08)JN021301 Dk/VN/ST/0970(09)JN021303 Dk/VN/BL(07)GU052526 85 65 35 Ck/VN/KH(2010)JN021304 Md/VN/BT342(06)EU124167 Dk/VN/CM/498A(06)EU124164 Dk/VN/AG(05)EF051514 16 Ck/VN/VL(05)EF057808 99 Dk/VN/DT680A(05)EU124134 A/VN(05)EF456799 45 Dk/VN/ST(05)EU124175 Ck/VN/DT171(04)DQ099759 Clade Ck/VN/TG023(04)DQ099758 46 Ck/VN/HD1(04)EF057807 65 53 Dk/VN/15(03)DQ497670 94 A/Hanoi/30408(05)AB239125 Ck/VN/8(05)CY016851-clade0 22 98 Dk/VN/MB2(07)JN021299 Md/VN/GL(04)EF051515 Ck/TL/CU/354(08)CY047457 29 A/VN/1203(04)EF541403 78 A/TL/NBL1(06)GQ466176 A/VN/1194(04)EF541402 Ck/Thailan/NIAH108(05)AB450565 Dk/HK/821(02)AY676033 Bh/Goose/Qinghai/67(05)DQ095623 51 100 Dk/VN/QT/801(2011)JN986881 99 99 Md/VN18159(09)JN055369 54 46 100 Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867 64 73 48 Dk/VN/QT/802(2011)JN986882 A/Hubei/1(2010)CY098758-clade2.3.2.1 Clade 2.3.2 Dk/VN/568(05)DQ320839clade2-3-2 Md/VN/1455(06)CY029535 Dk/China/E319/2(03)AY518362 Dk/VN/TMU021(09)DB/CY095695 44 35 85 A/FJ1(05)FJ492882 Dk/Laos/22(06-CY041043 Ck/FJ/584(06)DQ992831 86 Gs/GX1898(06)DQ993024 A/FJ1(07)FJ492883 Clade 2.3.4 Ck/VN/TMU004(08)HN/CY095680 34 A/VN/UT31394II(08)HM114593 53 A/VN/HN31242(07)EU294370 34 Dk/VN50(07)CY029711 41 Dk/VN/NA72(07)JN021305 46 66 Dk/VN/NA114(07)JN021306 Ck/VN/NCVD/8(03)EF541407-clade5 Swine/FJ/F1(01)AY747617-clade5 74 Clade Gs/VN/113(01)GU052105 99 85 Clade Gs/VN/324(01)GU052113 Dk/QX/22(01)AY585364-clade3 Gs/GD1(96)AF148678 44 A/HK156(97)AF046088 93 90 73 Dk/VN1(05)DQ366306 Clade A/HK/483(97)AF046097-clade0 Ck/VN/NCVD/093(08)FJ842480 100 Ck/VN17(08)FJ842478 Clade 0.01 Figure 3.3 Phylogenetic tree between the influenza A/H5N1 strains established based on the amino acid sequence of H5 using MEGA4.0 program (NJ - Neighbour Joining, testing 1000 bootstraps) Arrows indicate the strains isolated by us used for analysis in this study In the process of analyzing phylogenetic relationships based on nucleotide and amino acid composition of H5, we found some notable points: - The classic strain Gs-GD1-96 (AF148678) of clade was separately grouped together with the strains A-HK-483-97 (AF048097); A-HK156-97 (AF046088) and DkVN-1-05 (DQ866306) The strain DkVN-1-05 was yet isolated in Vietnam in 2005, but has been analyzed to be closely related to the H5N1 strains 13 isolated in humans in Hong Kong in 1997 (Hong Kong 97-like H5N1) This is a particular issue, because this strain emerged in Vietnam in 2005, but highly similar to the strains appeared in Hong Kong some years ago (1997) - The mixing of clades in Vietnam has been reported by a number of authors of Vietnam and overseas - Since its emergence in Vietnam there have been representative clades of avian influenza A/H5N1 virus circulating in Vietnam, that is clade 0, 1, (2.3.2; 2.3.4), 3, and In our analysis, the strains of Vietnam belonged to clades as the followings: clade 0: Dk-VN1-05 (DQ366306); clade 1: Appeared in 2004, 2005, 2006 - present in most provinces/cities nationwide, this clade still exists mainly causing severe disease in poultry in the Mekong River Delta; and recently, evolutionarily emerged as clade 1.1, mostly isolated after 2008; clade 2.3.2: This group of viruses have appeared since 2005 in Vietnam (eg, Dk-VN-568-05 (DQ320939)) and 2006 (eg Md-VN-1455-06 (CY029535)) belonged to genotype G virus Their origin is not assigned, particularly those strains appeared in 2009-2011, possibly from Qinghai lake (China) Two of our isolates (Dk/QT801-2011 and Dk/QT802-2011) collected in July, 2011, were identified belong to clade 2.3.2 but genetic differences were found from the strains of the same clade detected in Vietnam before clade 2.3.4: Started to appear in Vietnam from 2007 to 2009, this is the virus is highly pathogenic and potentially lethal high circulation mainly in the northern and central regions clade 3: Gs-VN-113-01 (GU052105) clade 5: Ck-VN-NCVD-8-03 (EF541407) clade 7: Has been found in Lang Son area (Ck-VN-NCVD093-08 (FJ842480)) and Dk-VN17-08-FJ842478 3.1.4 Determination of clades emerged in Vietnam In the period of circulation and causing disease in poultry, from 2003 to date it was revealed that there have been many clades evolved from the previously circulated clades Typically, clade introduced into Vietnam since 2003, clade 2.3.4 emerged after 2007, and recently, from 2009 clade 2.3.2.1 in the northern and clade 1.1 in the southern Vietnam To learn about the evolution of the A/H5N1 viruses, we have collected the representive strains for data, with a total of 28 H5 sequences representing clade and 1.1; and 32 representing the H5 sequence of 14 clade 2.3.2 and 2.3.4, including 14 strains of our study from 2004 to 2011 All these strains were put for comparative analysis and determination of clade based on standards/criteria by FAO / OIE / WHO launched recently (9/2011) 3.2 Determination of newly emerged clades in Vietnam 3.2.1 Study on formation of new clade 1.1 from clade Phylogenetic tree of clades of the A/H5N1 influenza viruses based on nucleotide is presented in Figure 3.4A and based on amino acids in Figure 3.4B 31 41 19 Ck/Thailand/CU/354(08)CY047457 A/Hanoi/30408(05)AB239125 51 77 Ck/VN/8(05)CY016851 99 Ck/VN/HD1(04)EF057807 15 A/Thailand/NBL1(06)GQ466176 Md/VN/GL(04)EF051515 13 74 99 92 Ck/Thailand/NIAH108(05)AB450565 Dk/VN/MB2(07) 100 Dk/VN/AG(05)EF051514 Ck/VN/VL(05)EF057808 83 Clade 54 50 Ck/VN/DT/171(04)DQ099759 Ck/VN/TG023(04)DQ099758 81 Dk/VN/SocTrang680C(05)EU124175 Ck/VN/LA636(05)EU124168 Ck/VN/29(07)CY029631 A/Cambodia/R0405050(07)FJ225472/clade 1.1 62 Md/VN/BenTre342(06)EU124167 Dk/VN/BL(07)GU052526 95 Dk/VN/CaMau/498A(06)EU124164 98 61 86 100 Dk/VN/0970(09) Ck/VN/KH(2010) Md/VN/OIE/559(2011)AB636524 60 53 49 Ck/VN/DT382(08) Ck/VN/KG88(08) 43 Ck/VN/TG023(04)DQ099758 Dk/VN/DT680A(05)EU124134 50 Dk/VN/ST(05)EU124175 Ck/VN/LA636(06)EU124168 Ck/VN/29(07)CY029631 69 A/Cambodia/R0405050(07)FJ225472/clade 1.1 Dk/VN/BacLieu(07)GU052526 50 29 Md/VN/BenTre/342(06)EU124167 Dk/VN/CaMau/498A(06)EU124164 Ck/VN/KG88(08) 45 Dk/VN/0970(09) 78 Md/VN/OIE/559(2011)AB636524 35 Ck/VN/TV98(08) 52 Ck/VN/KH(2010) 68 Ck/VN/DT382(08) 57 Clade 71 Dk/VN/DongThap680A(05)EU124134 100 A/Thailand/NBL1(06)GQ466176 A/VN/1194(04)EF541402/clade A/VN/1203(04)EF541403 22 Ck/Thailand/CU/354(08)CY047457 Md/VN/GL(04)EF051515 23 Dk/VN/MB2(07) 35 A/Hanoi/30408(05)AB239125 Ck/VN/8(05)CY016851 94 42 CkTL/NIAH108(05)AB450565 Ck/VN/HD1(04)EF057807 54 Dk/VN/AG(05)EF051514 45 Ck/VN/VL(05)EF057808 99 Ck/VN/DT171(04)DQ099759 30 A/VN/1194(04)EF541402/clade A/VN/1203(04)EF541403 Clade 1.1 Clade 1.1 Ck/VN/TV98(08) 0.002 0.005 A B Hình 3.4 A Phylogenetic tree between the influenza A/H5N1 strains established based on the amino acid sequence of H5 indicating clade and new clade 1.1 using MEGA4.0 program (NJ Neighbour Joining, testing 1000 bootstraps) Arrows indicate the strains isolated by us used for analysis in this study Strains (squared) are those recommended by WHO for vaccine development The results in Figure 3.4 is based on the nucleotide composition of five strains isolated by us during 2004-2007 (CkHD1-04; MdGL-04; CkVL05, and DkMB2 DkAG-05-07) were classified in the same clade with two strains identified by WHO to clade isolated in humans in Vietnam as A-VN-1194-04 (EF541402); A-VN-1203-04 (EF541403) To this clade, there are a number of strains such as AHN-30408-05 (AB239125) and some strains of Thailand in 2005: Ck-TL-NIAH108 (AB450565), A-2006-06 TL-NBL1 (GQ466176) 2008: Ck-TL-CU-354-08 (CY047457) Our 2008 isolates (CkDT382-08; CkKG88-08; CkTV98-08), 2009 (Dk0970-09) and 2010 (CkHK-2010) were identified in the new clade 1.1 of strains like OIE-Md-VN-559-2011 (AB636524) and human isolates of Cambodia (Cambodia-R0405050-2007 (FJ225472) and some strains of Vietnam isolated in 2006 and 2007 in Genbank 15 Similarly, when comparing amino acid composition to 14 strains isolated by us, including five strains of the 2004, 2005 and 2007., they were also identified to clade 1; and strains isolated in 2008, 2009, 2010 to clade 1.1 Thus, among our isolates mainly collected in the Southern Mekong Delta, as results, the strains isolated during 2004 - 2005 of clade 1, and the recent isolates (2008-2010) of the clade 1.1 recognized by FAO/WHO in 2011 3.2.2 Study on formation of clade 2.3.2 and 2.3.4 Results of analysis of our isolated strains including two strains in 2007 (DkNA72-07 and DkNA114-07) and strains in 2011 (DkQT801-2011 and DkQT802-2011) are presented in Figure.3.5A and Figure 3.5B Dk/VN/NA72(07) Dk/VN/NA114(07) Dk/HT/07/53(07)GU050428 41 Dk/VN/NA49(07)GU050421 45 A/VN/UT31388M1(07)HM114585 39 Md/VN/SL85(07)GU050491 40 A/VN/UT31413II(08)HM114609/clade 2.3.4.3 A/VN/UT31312II(08)HM114601 34 A/VN/HN31242(07)EU294369 44 A/VN/UT31312II(07)HM114577 98 94 Ck/VN/18082(09)/JN055370 100 Ck/VN/27310(09)JN055384 100 A/VN/UT31394II(08)HM114593 87 A/VN/HN31242(07)EU294369 A/VN/UT31312II(07)HM114577 22 A/VN/UT31413II(08)HM114609/clade 2.3.4.3 86 57 41 40 A/VN/UT31388M1(07)HM114585 57 Dk/VN/NA114(07) DkVN//HT/07/53(07)GU050428 53 33 54 60 97 2.3.4.3 90 0.005 A Dk/VN/NA49(07)GU050421 A/VN/UT31312II(08)HM114601 Ck/VN/18082(09)JN055370 Dk/VN/PT/07/48(07)GU050413 53 2.3.4.2 A/VN/HN31432M(08)HM114617 100 A/Anhui/1(05)/DQ371928/clade 2.3.4 2.3.4 100 Ck/Fujian/584(06)DQ992831 A/Guizhou/1(09)CY098737 2.3.4.1 A/Hunan/2(09)CY098751 100 Md/VN/1455(06)CY029535 2.3.2 Dk/VN/568(05)DQ320839/clade2.3.2 Md/VN/18159(09)JN055369 Little/Egret/HK/8863(07)CY036205 100 Dk/VN/QT801(2011) 100 61 Dk/VN/QT802(2011) 68 97 A/Hubei/1(2010)CY098758/clade 2.3.2.1 Crow/Bangladesh/11rs/1984/11(2011)JN795913 Dk/Laos/471(2010)CY098352 99 Dk/Fukushima/16(2011)AB629698 100 Dk/Hokkaido/WZ83(2010)AB612901 99 66 MalDk/Korea/W401(2011)JN202558 2.3.4.3 Dk/VN/NA72(07) 98 Ck/VN/27310(09)JN055384 A/VN/UT31394II(08)HM114593 59 DkVN//PT/07/48(07)GU050413 Ck/Bangladesh/11rs1984/30(2011)JN795924 61 A/Anhui/1(05)DQ371928 A/Guizhou/1(09)CY098737 2.3.4.1 A/Hunan/2(09)CY098751 100 2.3.4 Ck/Fujian/584(06)DQ992831 Ck/Bangladesh/11rs1984/30(2011)JN795924 34 33 49 Md/VN/SL85(07)GU050491 99 2.3.4.2 A/VN/HN31432M(08)HM114617 93 Md/VN/1455(06)CY029535 2.3.2 Dk/VN/568(05)DQ320839/clade2.3.2 70 Md/VN/18159(09)JN055369 Little/Egret/HK/8863(07)CY036205 100 88 100 Dk/VN/QT801(2011) Dk/VN/QT802(2011) 35 58 A/Hubei/1(2010)CY098758/clade2.3.2.1 Crow/Bangladesh/11rs/1984/11(2011)JN7959 2.3.2.1 Dk/Laos/471(2010)/CY098352 90 Dk/Fukushima/16(2011)AB629698 99 99 67 0.01 Dk/Hokkaido/WZ83(2010)AB612901 MalDk/Korea/W401(2011)JN202558 B Hình 3.5 Phylogenetic tree between the influenza A/H5N1 strains established based on the nucleotide sequence of H5 (panel A) and the amino acid sequence (panel B) indicating new clade 2.3.2.1 and new clade 2.3.4.3, using MEGA4.0 program (NJ - Neighbour Joining, testing 1000 bootstraps) Arrows indicate the strains isolated by us used for analysis in this study Strains (squared) are those recommended by WHO for vaccine development The results presented in Figure 3.5 showed that two strains of influenza A/H5N1 virus (DkNA72-07 and DkNA114-07) were grouped with strains of clade 2.3.4.3 comprising those isolated in poultry and humans in the years of 2007, 2008 and 2009, eg A-VN-UT31413II(2008) (GenBank: HM114609, clade 2.3.4.3) Two strains isolated in 2011 (DkQT801-2011 and DkQT802-2011) were of clade 2.3.2.1 and closely related to the strain A-Hubei-1-2010 isolated in human in China 2.3.2.1 16 It was clear, in Vietnam since the emergence of H5N1 viruses, there appeared the existence of many different clades, of which there has been recently clade 2.3.2.1 in 2011 In 14 strains of our study from 2004 to 2011, they were also grouped into clades, ie., clade 1, 1.1, 2.3.4.3 and 2.3.2.1 (Table 3.2) Table 3.2 Statistical summary of 14 strains of A/H5N1 influenza viruses identified based on H5 gene in period 2004-2011 in our study No 10 11 12 13 14 Abbreviations DkQT801-2011 DkQT802-2011 CkKH-2010 Dk0970-09 CkDT382-08 CkKG88-08 CkTV98-08 DkNA72-07 DkNA114-07 DkMB2-07 DkAG-05 CkVL-05 CkHD1-04 MdGL-04 Years isolated 2011 2011 2010 2009 2008 2008 2008 2007 2007 2007 2005 2005 2004 2004 Hosts Clade Genbank accession numbers duck duck chicken duck chicken chicken chicken duck duck duck duck chicken chicken muscovy duck 2.3.2.1 2.3.2.1 1.1 1.1 1.1 1.1 1.1 2.3.4.3 2.3.4.3 1 1 JN986881 JN986882 JN021304 JN021303 JN021302 JN021301 JN021301 JN021306 JN021305 JN021299 EF051514 EF057808 EF057807 EF051515 Some comments on characterization of the H5 gene Based on the results of analysis of nucleotide composition, amino acid similarity and the phylogenetic relationships, we have some comments as follows: The H5N1 strains isolated in Southeast Asia, have high identity of nucleotide (91-99%) and high similarity of amino acids (89-99%) between all the strains compared Among the Vietnamese H5N1 strains of Guangdong and Fujian sublineages, there have been no excessive levels of nucleotide mutations and amino acids beyond the level of identity/similarity within a subtype (hereby, H5 suntype), therefore, they are representative H5N1 of currently cirlulated strains in Vietnam 2- The H5N1 strains circulating in the South Vietnam, in the Mekong River Delta from 2006 to present, belong to clade 1.1 and clade appeared from 20042005 3- Two strains DkQT801-2011 and DkQT0802-2011, isolated in July, 2011, were classified to clade 2.3.2.1, currently causing outbreaks of A/H5N1 in several Northern provinces The analysis of H5 and N1 genes of these two strains of clade 17 2.3.2.1 would contribute to our understandings of the latest changes in the population of the A/H5N1 influenza viruses in our country 4- The H5 genes of 14 A/H5N1 viruses isolated in Vietnam in the period 20042011 were obtained and their H5 materials were stored in the cloning vectors giving rise for the selection of suitable strains in new generation vaccine development strategies 5- The glycosylation made differences between strains of clades due to the changes in site III: in clade it is NST; in clade 2.3.4 (2.3.4.3), it is NNT; in clade 2.3.2.1 no glycosylation at this site The new strains isolated in Quang Tri (of clade 2.3.2.1), glycosylation at site III is absent 6- Protease cleavage site sequences showed variation at position 341-345, in which in the Guangdong sublineage it was usually the motif (-RRRKK-), but in some strains of the Mekong River Delta this motif has changed to (-GRRKK-) In the Fujian sublineage it was (-RRRK-) All 14 strains of A/H5N1 viruses in our study there were these types of the motifs Analysis of phylogenetic relationship of the influenza A/H5N1 virus strains isolated in Southeast Asia, including strains in our study, we have come to conclusion that the virulent strains circulating to cause infections in Vietnam were of the same evolutionary origin uniquely derived from China 3.3 Amplification, sequencing and cloning of the N1 genes 3.3.1 Analysis of nucleotides and amino acid composition of neuraminidase subtype Recent studies on sequencing of genes/genomes of H5N1 circulating in Vietnam have shown that N1 genes of the A/H5N1 virus strains isolated before 1997 were from 1407 to 1410 nucleotides in size, while those isolated in recent 10 years to now (isolated after 1997 and between 2004 to date), due to deletionmutations of 57 or 60 nucleotides, the total remaining length is 1350 nucleotides, shorter than that in the isolates before 1997 The biological function of the polypeptide N1 depends on the first half of the amino acid sequence (N terminal), and changes in this polypeptide mostly occur in this area The kind of mutations (slippage-mediated deletion) were found to occur in the N1 genes through the different stages of evolution for the A/H5N1 18 viruses, and has been proven as a type of mutation which occured to generate N1 subtype of A/H5N1 viruses more pathogenic The results of studies also showed that H5N1 influenza viruses currently circulating to cause disease are undergoing strong variations in the genes/genomes to increase the virulence cause disease, and towards adaptive changes in the direction of transmission from poultry to humans The total length of all N1 genes in all strains in our study was 1350 nucleotides In these N1 genes, the initiative codon was ATG coding for the amino acid methionine (M) and the stop codons were TAG or TAA Results from analysis showed that the differences were mainly found in the first 80 amino acid polypeptide at the N terminal of N1 polypeptide The strains of the period 1997-2003 had the N1 gene of 1410 nucleotides in size, encoding 469 amino acids However, the strains isolated in humans in Hong Kong in 1997 (for example: A-HK-156-97) had removed 57 nucleotides (deletion-mutation), coding for 19 amino acids A strain isolated in 2005 (Dk-VN1-05) has been shown to have close relationship with the Hong Kong 97-like strains of H5N1 The strains emerged after the 1997, have evolved to delete 60-nucleotides in the way that nucleotides were retained in the 'end, and 12 nucleotides were deleted in the 5' end, compared with 57 nucleotides deleted in the strains of 1997) This mutation has made the way of creation of a slipage-deletion of nucleotides in the N1 gene during the evolutionary period After 2003, the length N1 has become stable, as seen in all 14 strains isolated by us having 1350 nucleotides in length, encoding 449 amino acids 3.3.2 Analysis of slippage-mediated deletion of the N1 genes through evolutionary period The N1 sequences for all 14 isolates in this study were fully obtained and used for comparative analysis of the nucleotide and amino acid composition The alignment of the N1 nucleotide sequence is presented in Figure 3.6 The strains are divided into three groups: I) the classical group containing strains isolated from 1996 to 2003; II) the group of intermediate Hong Kong strains in 1997; III) The group of circulating strains isolated from 2003 to present The results showed that strains of group (II) and group (III) had deletion mutation but the nature of mutations was different Deletion of nucleotides in group (II) occured 19 at positions 157-213 (57 nucleotide deletion compared with classical strains before 1997), while group (III) had 60 nucleotide deletion mutation in the position 145204 Twelve nucleotides (encoding amino acids) at the 5' had been removed and nucleotides (encoding amino acids) at the 3' had been retained in the N1 genes of the strains we have isolated (Figure 3.7) Display of a part of the N1 nucleotide sequence covering slipage-deletion A-HK-213-0 Dk-GD-22-0 Ck-HK-FY15 Gs-GD1-96A-HK-212-0 A-HK-482-9 A-HK156-97 Dk-VN1-05Dk-GD-12-0 CkHD1-04-E MdGL-04-EF A-Hatay-04 A-VN-HG207 A-VN-1194A-VN-1203Dk-ID-MS-0 Gs-TL-79-0 A-TL-676-0 DkAG-04-EF CkVL-05 Dk-VN-S654 DkCM1-06-F MdVN1455-0 DkHP208-06 Ck-NG-641DkNA72-07 DkNA114-07 DkMB2-08 A-VN31242DkDT9-07-F DkLao-07-C DkST41-08Dk19ATV-08 DkTV7B-08CkDT382-08 CkKG88-08 CkTV98-08 Dk0970-09 Ck-KH-2010 DkQT801-20 DkQT802-20 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : * 140 * 160 * 180 * 200 * 220 * 240 GGGAATCAACACCAGGCTGAACCATGCAATCAAAGCATTATTACTTATGAAAACAACACCTGGGTAAACCAGACATATGTCAACATCAGCAATACCAATTTTCTTACTGAGAAAGCTGTG .C A C .T A A T C A T A A T T C.C.C.A C -.T .TAC C.G CA T C.C.C.A C -.T .TAC C.G CA T C C.A C -.T .TAC C.G CA A . A . A . T A . A . T A AT T G AT T AT T CCC A.A . T GA.A . T A . T A . T A . T A G T A.T . T A A . A T .C G A . T A A A.T G T T A A.T G T T A A T A A.T G T T G C .A T T A T C C A G T .C A G T .C A G T .C A G T .C A A G T .C A GG A T .C C A G T C.C A G T .C AA A T C C A.T . T : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 240 240 240 240 240 183 183 183 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 I II III Figure 3.6 Location of the N1 nucleotide sequence where slipage-deletion occur in 14 strains studied (2004-2011) compared with strains in GenBank Notes: (I): strains of classical group, (II): Hong Kong intermediate groups in 1997; (III): Group of strains in circulation from 2003 to present; (.) Indicates identical nucleotides in the strains compared; the difference is represented by the letters Display of a part of the amino acid region involving deletion in N1 A-HK-213-0 Dk-GD-22-0 Ck-HK-FY15 Gs-GD1-96A-HK-212-0 A-HK-482-9 A-HK156-97 Dk-VN1-05Dk-GD-12-0 CkHD1-04-E MdGL-04-EF A-Hatay-04 A-VN-HG207 A-VN-1194A-VN-1203Dk-ID-MS-0 Gs-TL-79-0 A-TL-676-0 DkAG-04-EF CkVL-05 Dk-VN-S654 DkCM1-06-F MdVN1455-0 DkHP208-06 Ck-NG-641DkNA72-07 DkNA114-07 DkMB2-08 A-VN31242DkDT9-07-F DkLao-07-C DkST41-08Dk19ATV-08 DkTV7B-08CkDT382-08 CkKG88-08 CkTV98-08 Dk0970-09 Ck-KH-2010 DkQT801-20 DkQT802-20 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : * 20 * 40 * 60 * 80 * 100 * 120 MNPNQKITTIGSICMVIGIVSLMLQIGNIISIWVSHSIQTGNQHQAEPCNQSIITYENNTWVNQTYVNISNTNFLTEKAVASVTLAGNSSLCPISGWAVYSKDNGIRIGSKGDVFVIREPF .I .A P N .I .L I V I .H .I V I T V I.K.WHPN.P -I Y Q.A I S .I V I V I WHPN.P -I Y Q.A I S .I V I V I WHPN.P -I Y Q.A I S .I I H V .P I T M .H . .K N .S .I T M .H . .K N .S I .I T M .H . .K N .S .I T V M .H . .N .K N .S .I T M .H S L .K N .S .I T M .H S .K N .S .I M S.S P R H S K I T.M .L .H K .K N .S K I T.M .L .H QK .K N .S .I T V M .H . .K N .S .I T V M .H . .K N .S .I T V M .H . .K N .S .I T V M .H . .K N .S .I M I V R H S .I M .R .N R H S .I M R K .S R D I M N.V .I I R IH S .I M N.V .I I R IH S .I T M .H T .K N .S VI M N.V .I G I R IH S .I .AT V M TH T D. .KIV N .S .I V M .R S N R IH S .I .IT V M .H . P .AK N .S .I T V M .H . P .AK N .S .I T V M .H . P .AK N .S .I T V M .H . P .AK N .S .I T V M .H.E . P .AK N SS .I T V M N H .G. .R P .AK N .S .I T V M .H . P .AKF P N .S .I T V M .H . P .A N .S .I M T .R .N R H S .I M V R H S : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 121 121 121 121 121 102 102 102 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 101 I II Figure 3.7 Location of the N1 amino acid sequence where slipage-deletion occur in 14 strains studied (2004-2011) compared with strains in GenBank Notes: (I): strains of classical group, (II): Hong Kong intermediate groups in 1997; (III): Group of strains in circulation from 2003 to present; (.) Indicates identical amino acids in the strains compared; the difference is represented by the letters III 20 Nucleotide deletions in N1 are reviewed as follows: - The classical strains of influenza A/H5N1 (group i) had N1 gene with 1410 nucleotides in length - The strains in the intermediate group (group ii: isolated in Hong Kong in 1997) had deleted 57 nucleotides at positions 157-213 compared to the classical strains (5'-TCATTACTTACGAGAACAACACTTGGGTGAATCAAACATATGTTA ACATCAGCAAT-3 ') coding for 19 amino acids (IITYENNTWVNQTY NISN) - The circulating strains (group iii) isolated mainly after 2003, possess N1 sequences of 1350 nucleotides after undergone with 60 nucleotide mutations, at positions 145-204, coding for 20 amino acids (CNQSIITYENNTWVNQTYVN ) (Figure 3.6; Figure 3.7) The H5N1 strains isolated in Vietnam in our study, have N1 gene with 1350 nucleotides in length (449 amino acids) Characteristic glycosylation in N1 polypeptide In the polypeptide of the N1 of the recently isolated strains, glycosylation occured only in positions, ie NSS (position 88-90), NGT (position 146-148), and NGS (position 235-237) For the strains of the classical groups, there is one more glycoylation site of NNT (positions 58-60) (Figure 3.7) Results from analysis showed that the N1 gene in strains isolated after 2003 (may be called currently circulating strains) has not been required to maintain 60 nucleotides (for 20 amino acids) as the classical strains had, this evolution may be related to high pathogenicity of the newly emerged H5N1 strains today N1 gene sequences of 14 strains of the influenza A/H5N1 viruses isolated by us over the years from 2004 to 2011, as well as other strains in Genbank, have their length of 1350 bp, encoding 449 amino acids, reflecting appropriate evolution of N1 from 2003 to present The N-terminal of the polypeptide NA (N1) has much more changes than the other regions, reflected by a number of mutations and glycosylation positions than the C-terminal and tends to divide into groups by time However, upon being introduced into Vietnam so far, N1 in all the H5N1 isolates have only pointmutations which always occur in influenza A/H5N1 virus, no significant change in the current N1 genes as found in those isolates before 2003, which mainly focus on the N1 genes have already evolved 3.3.3 Phylogenetic relationships of the N1 strains of influenza A/H5N1 Analysis of the phylogenetic relationships based on nucleotide composition (Figure 3.8A), including 14 strains isolated by us from 2008 to 2011 period, that they are divided into five groups: 21 Group of strains isolated in 2011: consists of two strains (DkQT801 (2011) and DkQT802 (2011)), having close relationship with Dk-TB-07 (CY034698) and Dk-HP-208-06 (GU052504) strains, which have been identified to clade 2.3.2 based on H5 analysis N1-Nucleotide Ck/VN/KG88(08) Ck/VN/TV98(08) Dk/VN/0970(09) 46 Ck/VN/KH(2010) 62 Dk/VN/ST41(08)FJ812006 100 Dk/VN/19ATV(08)FJ812008 46 100 55 Dk/VN/TV7B(08)FJ812012 Ck/VN/DT382(08) 72 Dk/VN/CM1(06)FJ811997 48 Dk/VN/DT9(07)FJ811999 53 Ck/VN/VL(05) Clade 79 Dk/VN/S654(05)DQ321067 72 A/VN/HG207(05)DQ094292 21 Dk/VN/AG(04)EF057804 Md/VN/GL(04)EF057805 89 Ck/VN/HD1(04)EF057806 100 100 A/VN/Hatay(04)AJ867075 100 Gs/Thailand/79(04)AY651444 A/Thailand/676(05)DQ360836 51 Dk/VN/MB2(07) A/VN/1194(04)AY651445 38 77 A/VN/1203(04)AY651447 Ck/NG/641(06)DQ529296 Dk/ID/MS(04)AY651434 73 Dk/VN/NA72(07) Clade 2.3.4 100 Dk/VN/NA114(07) 96 A/VN31242(07)EU294372 Md/VN/1455(06)CY029537 90 Dk/VN/HP/208(06)GU052504 Dk/Laos/P0050(07)CY034698 Clade 2.3.2 Dk/VN/QT801(2011) 89 81 43 57 70 37 61 36 100 Dk/VN/QT802(2011) Gs/GD/1(96)AF144304/N1 71 Dk/GD/12(2000)AY585405 Clade 100 A/HK/213(03)AB212056 Nhóm cổ điển Dk/GD/22(02)AY585406 A/HK/212(03)AY575881 99 Ck/HK/FY150(01)AY221542 66 Dk/VN1(05)DQ366308 Clade A/HK/482(97)AF102656 Nhóm Hồng Kơng 100 (1997) 100 A/HK/156(97)AF046089 0.01 A N1-Amino acid Ck/VN/DT382(08) Dk/VN/0970(09) Ck/VN/KG88(08) 39 Ck/VN/TV98(08) 81 Dk/VN/ST41(08)FJ812006 95 Dk/VN/19ATV(08)FJ812008 53 85 63 Dk/VN/TV7B(08)FJ812012 Ck/VN/KH(2010) 49 Dk/VN/CM1(06)FJ811997 A/VN/HG207(05)DQ094292 27 Dk/VN/DT9(07)FJ811999 66 55 Ck/VN/VL(05) Dk/VN/S654(05)DQ321067 23 Dk/VN/AG(04)EF057804 Ck/VN/HD1(04)EF057806 87 A/VN/Hatay(04)AJ867075 31 Gs/Thailand/79(04)AY651444 39 A/Thailand/676(05)DQ360836 97 Md/VN/GL(04)EF057805 98 Dk/VN/MB2(07) A/VN/1194(04)AY651445 18 66 A/VN/1203(04)AY651447 Dk/ID/MS(04)AY651434 Md/VN/1455(06)CY029537 70 A/VN/31242(07)EU294372 17 Clade 2.3.4 72 Dk/VN/NA72(07) 100 Dk/VN/NA114(07) 77 Ck/NG/641(06)DQ529296 Dk/GD/12(2000)AY585405 19 Gs/GD1(96)AF144304 Clade 45 99 A/HK/213(03)AB212056 Nhóm cổ điển 20 A/HK/212(03)AY575881 Dk/GD/22(02)AY585406 97 Ck/HK/FY150(01)AY221542 65 Dk/VN/HP208(06)GU052504 81 Dk/Laos/P0050(07)CY034698 Clade 2.3.2 Dk/VN/QT801(2011) 56 Dk/VN/QT802(2011) 99 A/HK/482(97)AF102656 A/HK156(97)AF046089 100 Dk/VN1(05)DQ366308 90 42 27 0.01 Clade Clade Nhóm Hồng Kơng (1997) B Hình 3.8 Phylogenetic tree between the influenza A/H5N1 strains established based on the nucleotide sequence of N1 (panel A) and the amino acid sequence (panel B) using MEGA4.0 program (NJ - Neighbour Joining, testing 1000 bootstraps) Arrows indicate the strains isolated by us used for analysis in this study Some remarks about N1 of the influenza A/H5N1 virus isolated from 2004 to 2011: Group of the strains isolated in period of 2008-2009-2010: These include strains of our study as CkDT382-08; CkKG88 (08); CkTV98 (08); Dk0970 (09); CkKH-2010, grouped in a separate branch; and have close phylogenetic relationship with strains of DkST41-08 (FJ812006); Dk19ATV-08 (FJ812008); 22 DkTV7B-08 (FJ812012); DkCM1-06 (FJ811997); DkDT9-07 (FJ811999) of clade Group of the strains isolated in 2007: These include two strains (DkNA114-07 and DkNA72-07), located in the same group with a strain isolated in humans (A-VN31242-07 (EU294372)) These are the varieties of clade 2.3.4 Another strain (DkMB2-07) has close relationships with two strains of Vietnam (AVN-1194-04 (AY651445) and A-VN-1203-04 (AY651447)) and two strains of Thailand (Gs-TL -79-04 (AY651444) and A-TL-676-05 (DQ360836)) of clade 2006: Avian Influenza occurs scattered in Vietnam, we did not obtain any sample of the bird flu virus this year However, other authors have isolated two strains in Haiphong (DkHP208-06 (GU052504)) and in Ha Tay (Md1455-06 (CY029537)) of clade 2.3.2 with Dk-TB-07 (CY034698) and strain in Ca Mau (Dk-CM1-06 (FJ811997)) of clade Group of the strains isolated in 2005: These include two strains (CkVL-05 and DkAG-05) and our close relationship with strains Dk-VN-S654-05 (DQ321067); A-VN-HG207-05 (DQ094292) of clade Group of the strains isolated in 2004: These include two strains two strains of CkHD1-04 (EF057806) and MdGL-04 (EF057805) isolated by us, located on the same group with strains A-Hatay-04 (AJ867075), Gs-TL-79-04 (AY651444); ATL-676-05 (DQ360836) of clade Intermediate group of strains (Hong Kong 1997): including strains of 57 nucleotide delettion mutations in N1 gene The Dk-VN1-05 strain (DQ366398) isolated in Vietnam in 2005 is also placed in this group of clade Group of classical strains isolated in 1996: including strains of of the clade as Gs-GD 1-96 (AF144304) isolates in 1996 and is Dk-QD-2000 (AY585405) Analysis of relationships based on amino acid composition of N1 (Figure 3.8B), results are classified by the year as follows: 2011: strains (DkQT801-2011 and DkQT802-2011) with Dk-Lao-07 strains (CY034698) and Dk-HP-strain 208-06 (GU052504) belonged to clade 2.3.2 Years of 2008-2009-2010: strains of our study (CkDT382-08; CkKG8808; CkTV98-08; Dk0970-09; CkKH-2010), located in the group with Dk-ST-41-08 (FJ812006) Dk-19A-TV-08 (FJ812008) and Dk-TV-7B-08 (FJ812012) of clade Năm 2007: Two strain was isolated by us (DkNA114-07 and DkNA72-0707), is closely related to strains isolated in humans in Vietnam (A-VN-31242-07 23 (EU294372)), of clade 2.3 The DkMB2-07 strain, although isolated in 2007, but was placed in the same group of strains isolated in 2004 as CkHD1-04 (EF057806); MdGL-04 (EF057805); CkVL-05; DkAG-05 (EF057804); and Dk-VN-S654-05 (DQ321067); A-Hatay-04 (AJ867075), Gs-TL-79-04 (AY651444); A-TL-676-05 (DQ360836); A-VN-1194-04 (AY651445) and A-VN-1203-04 (AY651447) All were of clade Intermediate group of strains (Hong Kong 1997): includes A-HK-156-97 (AF046089); A-HK-482-97 (AF102656), grouped differently, in which strain-DkVN1 05 of Vietnam together in this group, of clade Classical Gs-QD-96: along with some isolates of 2001, 2002, 2003 (China) of clade The influenza A/H5N1 virus strains, from introduction in Vietnam so far, no significant change in N1 as differences appeared in the period 1996 - 1997 and 1997-2003, that the mutations are mainly focused on one N1 lineage of its evolution The results of this analysis is entirely consistent with the comments of the authors previously published CONCLUSION H5 antigenic genes of 14 strains studied, including: CkHD1 (04), MdGL (04), DkAG (05), CkVL (05), DkMB2 (07), DkNA72 (07), DkNA114 (07), CkDT382 (08), CkKG88 (08), CkTV98 (08), Dk0970 (09), CkKH (2010), (DkQT801 (2011), DkQT802 (2011) have been sequenced and cloned in the cloning vectors , depending on each strain, with a length of 1707 nucleotides (encoding 568 amino acids) or 1704 nucleotides (encoding 567 amino acids) N1 antigenic genes of 14 strains studied have also been sequenced and cloned in the cloning vectors Length of N1 gene is 1350 nucleotides (encoding 449 amino acids), due to mutation of 60 nucleotides (20 amino acids) removed in strains isolated after 2003, compared with 1410 bp or 1407 bp in the previous strains Identity level of nucleotide and homology of amino acid was 89 -99% in the 14 strains in this study compared with the strains isolated in Southeast Asia, depending on their clade 24 Forty strains isolated from 2004 to 2011 have been identified into four clades, namely: clade 1, clade 1.1, clade 2.3.2.1 and 2.3.4.3, where clade 1.1 (from 2008) and clade 2.3.2.1 (from 2011 ) are considered to be newly emerging in Vietnam The A/H5N1 viruses havea sites of glycosylation in the H5 polypeptide The change in position III (aa 170-172) is quite special, that it is NST in the strains of the Guangdong, and NNT in the Fujian lineage, while the recently isolated strains of clade 2.3.2.1 has been changed into DNA The connecting site (protease cleavage) between HA1-HA2 in H5 polypeptide has the motif (-RRRKK-) or (-RRRK-) or (-GRRKK-); and mutations in N1 showed that our strains during the period 2004-2011.reflected full characteristics of the important A/H5N1 populations circulating and causing disease in Vietnam SUGGESTIONS AND RECOMMENDATIONS Continue to investigate the influenza A/H5N1 viruses emerged in recent years belonging to new clades (clade 1.1 and 2.3.2.1), and to clone H5 and N1 genes of new clades of the different local isolates and different hosts in Vietnam in the next years and sequenced for comparative analysis with other strains Analysis should be done on more samples in order to study on the stability of the selected genes, and to select genetic resources as raw materials suitable for development of new-generation vaccines particularly in Vietnam Sequencing of all H5 and N1 genes and the entire genome of a new strain (clade 2.3.2.1 of the 2011 - 2012) for comparative analysis with other strains of the same clade in order to have database to evaluate genetic changes between the current influenza A/H5N1 viruses and making right orientation to immunity/immunization and prevention by vaccination  ... OVERVIEW 1.1 Outline of the influenza virus 1.1 .1 Influenza viruses and their classification 1.1 .2 Evolutionary history forming the different HPAI sublineages of the avian A/H5N1 viruses 1.1 .3 The. .. protein 1.3 .2 NA (neuraminidase) protein 1.4 The virulence determining factors 1.5 The ways of antigenic variation 1.5 .1 Antigenic drift 1.5 .2 Antigenic shrift 1.5 .3 Glycosylation 1.6 Introduction of. .. of the avian influenza 1.6 .1 History of the avian influenza 1.6 .2 Overall situation of the A/H5N1 avian influenza in the world 1.6 .3 Overall situation of the A/H5N1 avian influenza in Vietnam 1.6 .4