INTRODUCTION 1. The imperative research of the subject

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 vir

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INTRODUCTION

1 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

new/modified isolates (newly performed reassortants)

The NA antigenic polypepide is encoded by segment 6 This invloves in enzymatic process by cutting glycoside links at sialic acid molecules (N- acetylneuramic acid) at the receptor to release viral progeny during virus infection

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

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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

2 The objects of the theme

collected in different locations in Vietnam during 2004 to 2011.

2 Cloning H5 gene and N1 gene into the vectors for maintenance of the genetic materials for new generation vaccine development

identifying phylogenetic relationships with strains of influenza A/H5N1 in Vietnam and the world

3 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

4 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

4 Research objectives and outlines

The sampling strategy was to collect mucosal (exudate), tracheal, nasopharyngeal materials of the infected poultry that contained virulent avian

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

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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

5 Arrangement of the dissertation

The thesis consists of 99 pages, including the 3 page- Introduction; 35 page- Literature Overview; 11 page- Materials and Methods; 48 page- Results and Discussion; 2 page- Conclusions and Recommendations, and a List of publication;

12 page- References; 39-page Appendix

CHAPTER 1 LITERATURE OVERVIEW

avian A/H5N1 viruses

world and in Vietnam

avian A/H5N1 viruses

1.3.1 HA (hemagglutinin) protein

1.3.2 NA (neuraminidase) protein

1.5.1 Antigenic drift

1.5.2 Antigenic shrift

1.5.3 Glycosylation

1.6.1 History of the avian influenza

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1.6.8 Resistance of the avian influenza A/H5N1 virus

CHAPTER 2 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

into the vectors; Analysis of characteristics of the H5 and N1 antigen genes,

phylogenetic relationships in Vietnam and the world

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)

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

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analysis of phylogenetic relationships were done by using GENEDOC2.5 programs and MEGA4.0 on a PC computer.

CHAPTER 3 RESULTS AND DISCUSSION

3.1 Obtaining hemagglutinin sequences and cloning

3.1.1 Hemagglutinin subtype 5 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’

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 9 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

* 20 I * 40 II * 60 *

DkQT801-20 : MEKIVLLFASISLVKSDHICIGYHA NNS TEQVDTIMEK NVT VTHAQDILEKTHNGKLCDLNGVKPLILKDCSVAG : 75 DkQT802-20 : : 75

A-Hubei-1( : T : 75

CkKH-2010 : L.I Q D R R : 75

Dk0970(09 ) : IV Q D R R : 75

Dk-VN-TMU0 : L.IV R Q D R : 75

CkDT382(08 : IV Q T D R R : 75

CkKG88(08) : E IV Q D R R : 75

CkTV98(08) : IV R.Q D R R : 75

Ck-VN-TMU0 : L.I Q D R : 75

A-VN-UT313 : L.I Q E R : 75

CkTL-CU-35 : IV Q D R : 75

DkNA72-07 : L.I Q.Y R D R : 75

DkNA114-07 : L.I Q D R : 75

DkMB2-07 : IV Q D R : 75

DkBL(07)GU : IV Q D R : 75

DkVN50(07) : L.I Q D R : 75

A-VN(07)EU : L.I Q D R : 75

A-FJ1(07)F : L.IV Q D R : 75

DkCM(06)EU : IV Q D R : 75

MdBentre34 : IV Q D R : 75

A-TL(06)GQ : IV Q D.G R R : 75

A-VN(05)EF : IV Q D R : 75

A-FJ1(05)F : FL.IV Q D R : 75

DkAG-05 : IV Q D R : 75

CkVL-05(17 : IV Q D R : 75

DkVN1(05)D : L.TV Q R R : 75

CkHD1-04-E : IV Q G A.D R : 75

MdGL-04 : L IV Q R D R : 75

A-VN-1194- : IV Q D R : 75

A-VN-1203- : IV Q K D R : 75

A-HK156(97 : T L.TV Q R R : 75

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80 * 100 * 120 * 140 *

DkQT801-20 : WLLGNPMCDEFTNVPEWSYIVEKANPANGLCYPGNFNDYEELKHLLSRINHFEKIQIIPKDSWSDHEASLGVSAA : 150 DkQT802-20 : P G : 150

A-Hubei-1( : I D N : 150

CkKH-2010 : I V.D V S PS : 150

Dk0970(09 ) : I V.D V S PS : 150

Dk-VN-TMU0 : I D S S S S : 150 CkDT382(08 : I V.D V S PS : 150

CkKG88(08) : I D V S S : 150

CkTV98(08 ) : I V.D V S PS : 150

Ck-VN-TMU0 : I D L R S S S : 150 A-VN-UT313 : I D N S S : 150 CkTL-CU-35 : I V.D D S S S : 150 DkNA72-07 : I D S S SV : 150 DkNA114-07 : I D S S S : 150 DkMB2-07 : I V.D D S S S : 150 DkBL(07)GU : I V.D V S PS : 150

DkVN50(07) : I D S S S : 150 A-VN(07)EU : I D D S S S : 150 A-FJ1(07)F : I D S S : 150 DkCM(06)EU : I V.D V S PS : 150

MdBentre34 : I V.D V S PS : 150

A-TL(06)GQ : I V.D D S S S : 150 A-VN(05)EF : I V.D D S S : 150

A-FJ1(05)F : I D S S S : 150 DkAG-05 : I V V.D V S S S : 150 CkVL-05(17 : I V V.D V S S S : 150 DkVN1(05)D : I S D S N.D S S : 150 CkHD1-04-E : I V.D D S S S : 150 MdGL-04 : I V.D D S S S : 150 A-VN-1194- : I V.D D S S S : 150 A-VN-1203- : I V.D D S S S : 150 A-HK156(97 : I S D S N.D S S : 150 GsGD1(96)A : I S D D T PS N.D S S : 150 160 * III 180 IV * 200 * 220

DkQT801-20 : CPYQGSSSFFRNVVWLIKK DNA YPTIKKDY NNT NQEDLLILWGIHHPNDEAEQTRLYQNPTTYISIGTSTLNQRL : 225 DkQT802-20 : DNA : 225

A-Hubei-1( : K DNA G V : 225

CkKH-2010 : K NST RS D VM A K V : 225

Dk0970(09) : L.K NST RS VM A IK V : 225

Dk-VN-TMU0 : TP NNT RS V S AT IK V.V : 225

CkDT382(08 : K D NST RS D VM A K V : 225

CkKG88(08) : K NST RS D VM A AK I V : 225

CkTV98(08) : K NST RS D VM A K V : 225

Ck-VN-TMU0 : VP D NNT RS S A K V : 225

A-VN-UT313 : TP NNT RS S A K V : 225

CkTL-CU-35 : K NST RS V A K V : 225

DkNA72-07 : VP I NNT RS S A K V D : 225

DkNA114-07 : VP NNT RS S A K V D : 225

DkMB2-07 : K NST RS V A K V : 225

DkBL(07)GU : K NST RS VM A K V : 225

DkVN50(07) : VP NNT RS S A K V : 225

A-VN(07)EU : VP NNT RS S A K V : 225

A-FJ1(07)F : MP NNT RS S A K V : 225

DkCM(06)EU : K NST RS VI A K V : 225

MdBentre34 : K NST RS VM A K : 225

A-TL(06)GQ : K NST RS V W A K V : 225

A-VN(05)EF : K NST RS VM A K V : 225

A-FJ1(05)F : TP NNT RS S A K V : 225

DkAG-05 : K NST RS VI A IK V : 225

CkVL-05(17 : K NST RS VI AT IK V : 225

DkVN1(05)D : L.R NST RS V A K V : 225

CkHD1-04-E : K NST RS V A IK V : 225

MdGL-04 : K NST RS V A K V : 225

A-VN-1194- : K NST RS V A K V : 225

A-VN-1203- : K NST RS V A K V : 225

A-HK156(97 : L.R NST RS V A K V : 225

GsGD1(96)A : H.R NST RS V A K V : 225

* 240 * 260 * 280 * 300

DkQT801-20 : VPKIATRSKINGQSGRIDFFWTILKPNDAIHFESNGNFIAPEYAYKIVKKGDSTIMKSEVEYGNCNTRCQTPIGA : 300 DkQT802-20 : : 300

A-Hubei-1( : : 300

CkKH-2010 : T.R V ME N L K M : 300

Dk0970(09) : T.R V ME N N L K M : 300

Dk-VN-TMU0 : V M S N A S.K : 300

CkDT382(08 : T.R V ME F N L K M : 300

CkKG88(08) : T.R V ME N L K M : 300

CkTV98(08) : T.R V ME N L K M : 300

Ck-VN-TMU0 : V M N M K : 300

A-VN-UT313 : V M N A K : 300

CkTL-CU-35 : R V ME N L K M : 300

DkNA72-07 : V M N A K V : 300

DkNA114-07 : V M N A K : 300

DkMB2-07 : R V ME N L K M : 300

DkBL(07)GU : T.R V ME N L K V : 300

DkVN50(07) : V M N A K : 300

A-VN(07)EU : V M N A K : 300

A-FJ1(07)F : V M N A G K : 300

DkCM(06)EU : T.R V ME N L K M : 300

MdBentre34 : T.R V ME N L K M : 300

A-TL(06)GQ : R V ME N L K M : 300

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A-FJ1(05)F : V M N A.I K : 300

DkAG-05 : R V ME N L K M : 300

CkVL-05(17 : R V ME N GL K M : 300

DkVN1(05)D : E P.V ME N L K M : 300

CkHD1-04-E : R V ME N L K M : 300

MdGL-04 : R V ME N L K M : 300

A-VN-1194- : R V ME N L K M : 300

A-VN-1203- : R V ME N L K M : 300

A-HK156(97 : E P.V ME N L K M : 300

GsGD1(96)A : E P.V ME N A L K M : 300

HA1 HA2

V * 320 * 340 * 360 *

DkQT801-20 : I NSS MPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPQRE RRRK- RGLFGAIAGFIEGGWQGMVDGWYGYHHSND : 374 DkQT802-20 : R P RRRK- : 374

A-Hubei-1( : RRRK- E : 374 CkKH-2010 : R GRRRK E : 375 Dk0970(09) : TR GRRRK E : 375 Dk-VN-TMU0 : S L RRRK- E : 374 CkDT382(08 : L D R GRRRK E : 375 CkKG88(08) : L R GRRRK E : 375 CkTV98(08) : R GRRRK E : 375 Ck-VN-TMU0 : L RRRK- F E : 374 A-VN-UT313 : L RRRK- F E : 374 CkTL-CU-35 : R RRRKK E : 375 DkNA72-07 : L RRRK- S F E : 374 DkNA114-07 : L RRRK- F E : 374 DkMB2-07 : R RRRKK E : 375 DkBL(07)GU : R GRRRK E : 375 DkVN50(07) : L RRRK- F E : 374 A-VN(07)EU : L RRRK- F E : 374 A-FJ1(07)F : L RRRK- E : 374 DkCM(06)EU : R GRRRK E : 375 MdBentre34 : R GRRKK E : 375 A-TL(06)GQ : H R RRRKK E : 375 A-VN(05)EF : L R RRRKK E : 375 A-FJ1(05)F : L RRRK- E : 374 DkAG-05 : R K RRRKK E : 375 CkVL-05(17 : R K RRRKK E : 375 DkVN1(05)D : R A RRRKK : 375

CkHD1-04-E : R RRRKK E : 375 MdGL-04 : R RRRKK E : 375 A-VN-1194- : R RRRKK E : 375 A-VN-1203- : R RRRKK E : 375 A-HK156(97 : R T RRRKK E : 375 GsGD1(96)A : R T RRRKK N E : 375 380 * 400 * 420 * 440 *

DkQT801-20 : QGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMEN : 449 DkQT802-20 : R : 449

A-Hubei-1( : : 449

CkKH-2010 : : 450

Dk0970(09) : : 450

Dk-VN-TMU0 : : 449

CkDT382(08 : : 450

CkKG88(08) : : 450

CkTV98(08) : S : 450

Ck-VN-TMU0 : : 449

A-VN-UT313 : : 449

CkTL-CU-35 : N : 450

DkNA72-07 : : 449

DkNA114-07 : : 449

DkMB2-07 : : 450

DkBL(07)GU : : 450

DkVN50(07) : : 449

A-VN(07)EU : : 449

A-FJ1(07)F : : 449

DkCM(06)EU : : 450

MdBentre34 : : 450

A-TL(06)GQ : H : 450

A-VN(05)EF : : 450

A-FJ1(05)F : : 449

DkAG-05 : : 450

CkVL-05(17 : : 450

DkVN1(05)D : PPNQK LN.F.I FT IT : 450

CkHD1-04-E : : 450

MdGL-04 : : 450

A-VN-1194- : : 450

A-VN-1203- : : 450

A-HK156(97 : N : 450

GsGD1(96)A : Q : 450

460 * 480 * 500 VI * 520

DkQT801-20 : ERTLDFHDSNVRNLYDKVRLQLKDNAKELGNGCFEFYHKCNNECMESVR NGT YDYPQYSEEARLKREEISGVKME : 524 DkQT802-20 : : 524

A-Hubei-1( : D L : 524

CkKH-2010 : K R D L : 525

Dk0970(09) : K D L : 525

Dk-VN-TMU0 : K R D L : 524

CkDT382(08 : G K R D L : 525

CkKG88(08) : K R D.K L : 525

CkTV98(08) : K R D I L : 525

Ck-VN-TMU0 : K R D L : 524

A-VN-UT313 : K R DD K L : 524

CkTL-CU-35 : K R D L : 525

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DkNA114-07 : K R D L : 524

DkMB2-07 : K R D L : 525

DkBL(07)GU : K R D L : 525

DkVN50(07) : K R D L : 524

A-VN(07)EU : K R D L : 524

A-FJ1(07)F : K R DD L : 524

DkCM(06)EU : K R D L : 525

MdBentre34 : K R D L : 525

A-TL(06)GQ : K R DD L : 525

A-VN(05)EF : K R D L : 525

A-FJ1(05)F : K R D L : 524

DkAG-05 : K R D L : 525

CkVL-05(17 : K R D L : 525

DkVN1(05)D : K R D K V N L : 525

CkHD1-04-E : K R D L : 525

MdGL-04 : K R D L : 525

A-VN-1194- : K R D L : 525

A-VN-1203- : K R D L : 525

A-HK156(97 : K R D K N L : 525

GsGD1(96)A : KH R D K N L : 525

* 540 * 560 VII

DkQT801-20 : SIGIYQILSIYSTVASSLVLAIMMAGLSLWMCS NGS LQCRICI- : 567 DkQT802-20 : - : 567

A-Hubei-1( : - : 567

CkKH-2010 : V A V - : 568

Dk0970(09) : V A V - : 568

Dk-VN-TMU0 : T V - : 567

CkDT382(08 : V A LV - : 568

CkKG88(08) : V.V A V - : 568

CkTV98(08 ) : V A I - : 568

Ck-VN-TMU0 : T A V - : 567

A-VN-UT313 : T A V - : 567

CkTL-CU-35 : A V - : 568

DkNA72-07 : T A V - : 567

DkNA114-07 : T A V - : 567

DkMB2-07 : A V - : 568

DkBL(07)GU : A V - : 568

DkVN50(07) : T A V - : 567

A-VN(07)EU : T A V - : 567

A-FJ1(07)F : T A V - : 567

DkCM(06)EU : A V - : 568

MdBentre34 : A V - : 568

A-TL(06)GQ : A V - : 568

A-VN(05)EF : A V - : 568

A-FJ1(05)F : T A V - : 567

DkAG-05 : A I - : 568

CkVL-05(17 : A I - : 568

DkVN1(05)D : M.T A V - : 568

CkHD1-04-E : A V - : 568

MdGL-04 : A V H - : 568

A-VN-1194- : A V - : 568

A-VN-1203- : A V - : 568

A-HK156(97 : M.T A V - : 568

GsGD1(96)A : M.T A V - : 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 receptor-binding 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 receptor-binding 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, 4 strains of

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China, 2 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 7 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 6 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

Protease cleavage site

Recept or- bindin

g site

Epitope sites

Glycosyl ation position

A/chicken/Vietnam/TMU004/2008 Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT

A/Vietnam/UT31394II/2008 Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGTP NNT

A/Duck/Vietnam/NA72/ 2007* Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT A/Duck/Vietnam/NA114/2007* Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT

A/duck/Vietnam/50/2007 Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT

A/Vietnam/HN31242/2007 Vietnam 2.3.4.3 LRERRRK-R QSG R ANPA DHEASS PYQGVP NNT A/Fujian/1/2007 China 2.3.4 LRERRRK-R QSG R ANPA DHEASL PYQGMP NNT

A/Fujian/1/2005 China 2.3.4 LRERRRK-R QSG R ANPA DHEASS PYQGTP NNT

A/Chicken/Vietnam/KH/ 2010* Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/0970/ 2009* Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/DT382/2008* Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/Chicken/Vietnam/KG88/2008* Vietnam 1.1 QREGRRKKR QSG R ANPA SHEASL PYQGKS NST A/Ck/Vietnam/TV98/2008 * Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/duck/BacLieu/07-09/2007 Vietnam 1.1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/Ca Mau/498A/2006 Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/Muscovy Duck/Vietnam/ BenTre/

342/2006 Vietnam 1.1 QREGRRKKR QSG R ANPV SHEASL PYQGKS NST A/Chicken/Thailand/CU-354/2008 Thailand 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/MB2/ 2007* Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Thailand/NBL1/2006 Thailand 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Vietnam/CL2009/2005 Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/AG/2005* Vietnam 1 Q ERRRKKR QSG R VNPV SHEASL PYQGKS NST A/Chicken/Vietnam/VL/ 2005* Vietnam 1 Q ERRRKKR QSG R VNPV SHEASL PYQGKS NST A/Chicken/Vietnam/HD1/2004* Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Muscovy Duck/Vietnam/GL/2004* Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Vietnam/1194/2004 Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Vietnam/1203/2004 Vietnam 1 QRERRRKKR QSG R ANPV SHEASL PYQGKS NST A/Duck/Vietnam/1/2005 Vietnam 0 QRERRRKKR QSG R ASPA NHDASS PYLGRS NST A/HongKong/156/1997 Hongkong 0 QRERRRKKR QSG R ASPA NHDASS PYLGRS NSA

A/Goose/Guangdong/1/1996 China 0 QRERRRKKR QSG R ASPA NHDASS PYHGRS NSA

Note: The symbol (*) are the strains isolated and identified in this study

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- 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 0 (Gs-GD1-96; A- KH156-97 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 1 (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-VN- 1194-04) 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-CN- GD1-96(H5N1) and H5N2 vaccine strains are the original a-Turkey-ENG-N28- 73(H5N2)) Currently, two strains of A-VN-1194-2004(H5N1) and A-VN-1203- 2004(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, 4 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

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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)

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; MdGL-

04, were grouped in clade 1 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.

Clade 2.3.2

Ck/VN/TV98(08)JN021300 Ck/VN/KG88(08)JN021301 Dk/VN/ST0970(09)JN021303 Ck/VN/KH(2010)JN021304

Dk/VN/BL(07)GU052526 Dk/VN/CM498A(06)EU124164 Md/VN/BT342(06)EU124167 Dk/VN/ST(05)EU124175

A/VN(05)EF456799 Dk/VN/DT680A(05)U124134

Dk/VN/AG(05)EF051514 Ck/VN/VL(05)EF057808

Ck/VN/DT171(04)DQ099759 Ck/VN/TG023(04)DQ099758

Dk/VN/MB2(07)JN021299 Ck/VN/HD1(04)EF057807

Dk/VN15(03)DQ497670 A/Thailand/NBL1(06)GQ466176 A/VN/Hanoi30408(05)AB239125 Ck/VN8(05)CY016851-clade0 Ck/Thailand/CU354(08)CY047457 A/VN1194(04)EF541402 A/VN1203(04)EF541403

Md/VN/GL(04)EF051515

Ck/Thailand/NIAH108(05)AB450565 Dk/HK821(02)AY676033

Bh/Goose/Qinghai/67(05)DQ095623

Dk/VN/QT801(2011)JN986881 Dk/VN/QT802(2011)JN986882

A/Hubei/1(2010)CY098758-clade2.3.2.1 Md/VN18159(09)JN055369

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/VN568(05)DQ320839 Md/VN1455(06)CY029535 Dk/China/E319/2(03)AY518362

Dk/VN/TMU021(09)DB-CY095695 A/FJ1(05)FJ492882

Ck/FJ584(06)DQ992831 A/FJ1(07)FJ492883

Gs/GX1898(06)DQ993024 Dk/Laos22(06)CY041043

A/VN/UT31394II(08)HM114593 Ck/VN/TMU004(08)HN-CY095680

Dk/VN/NA72(07)JN021305 Dk/VN/NA114(07)JN021306

Dk/VN50(07)CY029711 A/VN/HN31242(07)EU294370 Ck/VN/NCVD/8(03)EF541407-clade5 Swine/FJ/F1(01)AY747617-clade5

Dk/VN1(05)DQ366306 A/HK483(97)AF046097-clade0 A/HK156(97)AF046088 Gs/GD1(96)AF148678

Ck/VN/NCVD/093(08)FJ842480 Ck/VN17(08)FJ842478 Dk/QX/22(01)AY585364-clade3

Gs/VN113(01)GU052105 Gs/VN/324(01)GU052113

100

100 100

100

99

34 58 100

100 100

100

91

43 46 47 100

100

63

93 100

97

98

54 34

36

45 42 99

61 94

100

99

98

58 66 99

89 57 79

83

43 100 45

38

41 20

Clade 7 Clade 3

Clade 2.3.2

A/VN(05)EF456799 Dk/VN/DT680A(05)U124134

Md/VN/GL(04)EF051515

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Ck/FJ584(06)DQ992831 A/FJ1(07)FJ492883

Gs/GX1898(06)DQ993024 Dk/Laos22(06)CY041043

Gs/VN113(01)GU052105 Gs/VN/324(01)GU052113

100

100 100

100

99

34 58 100

100 100

100

91

43 46 47 100

100

63

93 100

97

98

54 34

36

45 42 99

61 94

100

99

98

58 66 99

89 57 79

83

43 100 45

38

41 20

Clade 7 Clade 3

A/VN(05)EF456799 Dk/VN/DT680A(05)U124134

Md/VN/GL(04)EF051515

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Ck/FJ584(06)DQ992831 A/FJ1(07)FJ492883

Gs/GX1898(06)DQ993024 Dk/Laos22(06)CY041043

Gs/VN113(01)GU052105 Gs/VN/324(01)GU052113

100

100 100

100

99

34 58 100

100 100

100

91

43 46 47 100

100

63

93 100

97

98

54 34

36

45 42 99

61 94

100

99

98

58 66 99

89 57 79

83

43 100 45

38

41 20

Clade 7 Clade 3

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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 0 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

Clade 2.3.2

H5 AMINO ACID Ck/VN/KH(2010)JN021304

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 Md/VN/BT342(06)EU124167 Dk/VN/CM/498A(06)EU124164

Dk/VN/DT680A(05)EU124134 A/VN(05)EF456799 Dk/VN/ST(05)EU124175 Ck/VN/DT171(04)DQ099759 Ck/VN/TG023(04)DQ099758

Ck/VN/HD1(04)EF057807

Dk/VN/15(03)DQ497670 A/Hanoi/30408(05)AB239125 Ck/VN/8(05)CY016851-clade0

Dk/VN/MB2(07)JN021299 Md/VN/GL(04)EF051515

Ck/TL/CU/354(08)CY047457 A/VN/1203(04)EF541403 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

Dk/VN/QT/801(2011)JN986881 Dk/VN/QT/802(2011)JN986882

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/VN/568(05)DQ320839clade2-3-2 Md/VN/1455(06)CY029535

Dk/China/E319/2(03)AY518362

Dk/VN/TMU021(09)DB/CY095695 A/FJ1(05)FJ492882

Dk/Laos/22(06-CY041043 Ck/FJ/584(06)DQ992831 Gs/GX1898(06)DQ993024

A/FJ1(07)FJ492883 Ck/VN/TMU004(08)HN/CY095680 A/VN/UT31394II(08)HM114593 A/VN/HN31242(07)EU294370 Dk/VN50(07)CY029711

Ck/VN/NCVD/8(03)EF541407-clade5 Swine/FJ/F1(01)AY747617-clade5 Gs/VN/113(01)GU052105 Gs/VN/324(01)GU052113 Dk/QX/22(01)AY585364-clade3 Gs/GD1(96)AF148678 A/HK156(97)AF046088

Dk/VN1(05)DQ366306 A/HK/483(97)AF046097-clade0

Ck/VN/NCVD/093(08)FJ842480 Ck/VN17(08)FJ842478

100

100 99

85

41 34

73

53

85

34 86

54 64 48

44

44 74

35 46 51 78

99

35 41 74

65

31 85

94 35

98 53

16

46 45

65

22 29

H5 AMINO ACID Ck/VN/KH(2010)JN021304

Ck/VN/DT382(08)JN021302 Ck/VN/TV98(08)JN021300 Ck/VN/KG88(08)JN021301 Dk/VN/ST/0970(09)JN021303

Ck/VN/HD1(04)EF057807

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/China/E319/2(03)AY518362

100

100 99

85

41 34

73

53

85

34 86

54 64 48

44

44 74

35 46 51 78

99

35 41 74

65

31 85

94 35

98 53

16

46 45

65

22 29

Ck/VN/HD1(04)EF057807

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/China/E319/2(03)AY518362

100

100 99

85

41 34

73

53

85

34 86

54 64 48

44

44 74

35 46 51 78

99

35 41 74

65

31 85

94 35

98 53

16

46 45

65

22 29

Ck/VN/HD1(04)EF057807

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/China/E319/2(03)AY518362

100

100 99

85

41 34

73

53

85

34 86

54 64 48

44

Ck/VN/KH(2010)JN021304 Ck/VN/DT382(08)JN021302 Ck/VN/TV98(08)JN021300 Ck/VN/KG88(08)JN021301 Dk/VN/ST/0970(09)JN021303

Ck/VN/HD1(04)EF057807

Dk/VN27373(09)JN055388 Ck/VN10(05)CY016867

Dk/China/E319/2(03)AY518362

100

100 99

85

41 34

73

53

85

34 86

54 64 48

44

44 74

35 46 51 78

99

35 41 74

65

31 85

94 35

98 53

16

46 45

65

22 29

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