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

Clade 1, 2 and 7 strains belong to Z, V and G genotypes of the A/H5N1 virus, are widespeared circulated virus groups, causeA/H5N1 oubreaks in poultry specieses and infected to human, inV

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 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

- Library Hanoi Medical University

- Library Central Health information

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.

Huong, Dang Thi Ngoc Dung, Le Thanh Hoa(2011), “Molecular properties of polymerase PB1 gene ofDkNA72 and DkNA14 isolates of the Fujian sublineeage

A/H5N1 influenza virus isolated in Vietnam”, Journal of

Military Pharmaco - medicine, 36(1), pp 36-41.

(2012), “Molecular properties of the polymerase complex ofthe A/H5N1 clade 2.3.2.1 isolated from ducks in Quang Tri

province in 2011”, Journal of Vietnam Medicine, 396(1), pp.

50-56

ABREVIATIONS IN THE DISSERTATION

DNA Deoxyribonucleic acid

dNTP Deoxy Nucleotide Triphosphate

ddNTP dideoxy Nucleotide Triphosphate

FAO Food and Agricultural Organization

OIE Office International des Epizooties

PA Polymerase acidic protein

PB1 Polymerase basic protein 1

PB2 Polymerase basic protein 2

RT-PCR Revertranscription Polymerase Chain Reaction RNA Ribonucleic acid

RNP Ribonucleoprotein

(-) ssRNA Negative single-strand Ribonucleic AcidWHO World Health Organization

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 tochange them infected and transmition abilities to human and manyanimal specieses, caused A influenza pandemics in human in history.PB2, PB1 and PA genes encoding PB2, PB1 and PA polymeraseproteins, are subunits of enzym polymerase complex This complex

is importal role to decide adapted transcription of the virus in thehost specieses In addition, PB1 gen contants 2 open reading frame(ORF), encoding PB1-F2 and PB1-N40 proteins, that join in thevirulence expresion of the A influenza virus in the infected host.Since 2003, A/H5N1 highly virulent virus cause many A/H5N1oubreaks in the poultry, infected and cause serious acute flu with thehigh mortality rate (over 60%) in the human population In there,Vietnam is one of countries, that have A/H5N1 avian influenzaoubreaks in 2003 and repeated in many provinces and cities, withover 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, causeA/H5N1 oubreaks in poultry specieses and infected to human, inVietnam 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, havemuch different various of the H5 antigenic and high virulence, incomparision virus strains in the past, and complicate the A/H5N1influenza oubreaks prevention

The genetically variation of PB2, PB1 and PA polymerase helpA/H5N1 influenza virus to adapt transcription in infected cells,decides easilier transmition of virus from human to human, and

increases virulence in the body of human At present, this is one ofproblems that being interested in supervising and studying A/H5N1virus, pathogenic in domestic poultry and human The geneticallydata of PB2, PB1 and PA provide a scientific basic to forecast earlymolecular 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 homologyrate of nucleotide and amino acid of PB2, PB1 and PA genes of someA/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/H5N1strains 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 containedA/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 2pages, Chapter 1 – Overview is 30 pages, Chapter 2 – Subjects andmenthod is 20 pages, Chapter 3 – Results of research is 35 pages,Chapter 4 – Discussion is 26 pages, Conclusion is 2 pages andRecommendations is 1 page List of publication is 1 page,References 13 pages and Appendix is 39 pages In dissertation: 29tables, 26 figures The dissertation has 118 references, including: 14documents of Vietnamese, 104 documnents of English and 8 web pages

Chapter 1 OVERVIEW

1.1 BIOLOGICAL CHARACTERISTICS OF THE A INFLUENZA VIRUS1.1.1 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 genomethe A influenza virus

1.1.4 Structural and fuctional characteristics of PB2, PB1 and PA genes1.1.5 The polymerase complex of the A influenza virus

1.1.6 Genetically variation characteristic of genes and thegenome A influeza virus

1.2 A INFLUENZA PANDEMIC AND VARIATIONAL CHARACTERISTIC

OF PB2, PB1 AND PA GENES OF THE HUMAN AINFLUENZA VIRUS

1.2.1 A influenza pandemics in the past

1.2.2 Variational characteristic of PB2, PB1 and PA of thehuman 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 GENESRELATED VIRULENCE AND TRANSMISSION OF THEA/H5N1 INFLUENZA VIRUS IN HUMAN

1.4.1 In the world

1.4.2 In Vietnam

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 and1.1, 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 andchickens were died in A/H5N1 avian influenza oubreaks, that occurs

at some location in Vietnam during 2007 – 2011, to receivenucleotide and amino acid sequences of PB2, PB1 and PA genes ofthe genome of these virus strains

- All of 6 material samples content corresponding A/H5N1influenza virus strains, based sequencing and definning results of H5gene, that was studied and publicated by authors: Nguyen Thi BichNga and Le Thanh Hoa (2012) Material samples and A/H5N1 virusstrain in them, is abbreviation sign and full name as the internationalnomenclature in this study (Table 2.1)

Table 2.1 List of six A/H5N1 influenza virus strains in this study

Number VIRUS STRAINS SAMPLES /

SIGN

INTERNATIONAL NOMENCLATURE Year

isolated

CLADE H5

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.

+ PB2, PB1 and PA genes of 6 A/H5N1 strains in this study, werecollected, analyzed and compared genetically variation withcorresponding genes of 25 A/H5N1 virus strains, belong to 4 virusgroups: 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 virusstrains, was obtained from online database of Genbank, that haveclade H5 and virus genotype was defined and publicated indocuments 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 biologicaltechnique, 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/H5N1influenza 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 Bioneercompany (South Korean

PROCESSING AND OBTAINING THE NUCLEOTIDE SEQUENCE OF PB2, PB1 AND PA GENES

(USING BIO-INFORMATICS PROGRAM S TO OBTAINING THE NUCLEOTIDE AND AMINO ACID SEQUENCES)

OBTAINING DNA OF PB2, PB1 AND PA GENES BY PCR TECHNIQUE

( USING SPECIFIC PRIME PAIRS TO OBTAIN EVERY INDIVIDUAL GENE )

CLONING AND OBTAINING THE DNA RECOMBINANT PLASMID

OF PB2, PB1 AND PA GENES

SEQUENCING DNA OF PB2, PB1 AND PA GENES FROM RECOMBINANT PLASMID

(OBTAINING THE NUCLEOTIDE SEQUENCE OF PB2, PB1 AND PA GENES)

COMPARISING, ANALYSING GENETICALLY VARIATION AND DETERMINING PHYLOGENETIC RELATIONSHIPS OF PB2, PB1 AND PA GENES

(USING GENEDOC 2.5 AND MEGA 4.1PROGRAM S)

VIRAL RNA EXTRACTION AND TRANSCRIPTION INTO cDNA OF THE GENOME VIRUS

FROM MATERIAL SAMPLES

(USING RANDOM HEXAME AND 468F PRIMERS DESIGNED FOR cDNA TRANSCRIPTION)

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 andhexamer primes, according to protocol provided by using theMaxima™ Universal First Strand cDNA Synthesis Kit (Fermentas),

- Using the PCR technique to obtain DNA sequences of PB2, PB1and PA genes in the genome virus, with viral cDNA and specificprimepairs, 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 usingthe AccuPrep® Gel purification Kit (Bioneer)

2.2.5 Electrophoresis nucleic acid

2.2.6 Cloning DNA

Purified DNA products of PB2, PB1 and PA genes were clonedinto 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 DyeTerminator 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 wasprocessed by using SeqEd v1.03 and MacVector 8.2 (Accelrys Inc.)programs on the Macintosh computer

Analysing, comparing of nucleotide and amino acid sequenceswas done by the GENEDOC 2.5 program Analysing of phylogeneticrelationships was done by using the MEGA4.1 program on thepersonal computer

2.3 THE ETHICAL ASPECTS OF THE RESEARCH

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 wereobtained, after steps: viral RNA extraction, RT-PCR two steps,sequencing, processing and comparison with nucleotide sequences ofthe corresponding genes of the A/H5N1 virus, that stored in onlinedata of the Genbank

Analysis results:

- PB2, PB1 and PA polymerase genes of 6 A/H5N1 strains in thisstudy, contain 2.280, 2.274 and 2.151 nucleotides, respectively Oneafter 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 thePB1-F2 open reading frame (including 273 nucleotides, encode 90amino acids) and the PB1-N40 open reading frame (including 757nucleotides, encode 718 amino acids)

- The nucleotide sequence of PB2, PB1 and PA genes wereobtained after sequencing, are 97% - 99% homology rate in 96% -100% comparison, with corresponding gene sequences of theA/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 withcorresponding sequences of 19 A/H5N1 strains belong to 4 virusgroups: clade 2.3.2.1, 2.3.4.3, 1 và 1.1

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 aminoacids, as nucleotide and amino acid amount as compared with thisgene of above 19 virus strains

- Apart from many separate different positions, there are 110positions specific difference of nucleotide in PB2 sequence, between

6 strains of studying and 19 strains represented 4 comparative virusgroups However, have only above 18/110 nucleotide differentpositions lead to change amino acid in the PB2 protein sequence

- Six A/H5N1 strains of studying and A/H5N1 virus strainsisolated from domestic poultry in 4 comparative virus groups, arePB2 protein sequence conseved amino acid glutamic acid at the 627thposition (E627) and aspartic acid at the 701th position (D701)

- In particular, PB2 nucleotide sequence of 7 A/H5N1 strainsisolated from patients of clade 1 and 2.3.4.3 virus groups, have amutation to change nucleotide (A↔C) at the 1897th position, leads tochange amino acid at the 627th (E627K) position in the PB2 protein,

in comparison with corresponding sequence of virus strains isolatedfrom 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/H5N1virus strains were compared in this study (Table 3.1)

- These rate of the PB2 gene between 6 A/H5N1 virus strains ofstudying and represented strains in the 4 virus groups, isolated inChina, Thailand, Cambodia and Lao, are 96% – 99% of thenucleotide and 98% – 100% of the amino acid (Table 3.1)

Bảng 3.1 The homology rate (%) of nucleotide and amino acid of the PB2 gene Numberica

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;