UNIVERSITI PUTRA MALAYSIA

CLONING AND EXPRESSION OF NUCLEOCAPSID PROTEIN OF NEWCASTLE DISEASE VIRUS FUSED WITH VP5 GENE OF

INFECTIOUS BURSAL DISEASE VIRUS

SHAHERNY ZAID

FBSB 2007 16

CLONING AND EXPRESSION OF NUCLEOCAPSID PROTEIN OF NEWCASTLE DISEASE VIRUS FUSED WITH VP5 GENE OF

INFECTIOUS BURSAL DISEASE VIRUS

SHAHERNY ZAID

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2007

CLONING AND EXPRESSION OF NUCLEOCAPSID PROTEIN OF NEWCASTLE DISEASE VIRUS FUSED WITH VP5 GENE OF INFECTIOUS

BURSAL DISEASE VIRUS

By

SHAHERNY ZAID

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of

Science

June 2007

This thesis is especially dedicated to:

My Mom and family, who are infinitely very precious to me.

And

to my beloved, Zulkefley Othman and my lovely daughter, Nurqistina Batrisya who have colored my life with joy and happiness.

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TABLE OF CONTENTS Page DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEDGEMENTS vii APPROVAL viii DECLARATION xi LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 2.1 IBDV and the disease 5 2.2 History and epidemiology 5 2.3 Taxonomic classification 6 2.4 Virus strain 8 2.5 Transmission 10 2.6 Pathogenesis 10 2.7 Immunosupression 12 2.8 Economic impact 14 2.9 Prevention and control 15 2.10 IBDV 16 2.10.1 Virus structure and genome organization 16 2.10.2 Viral proteins 18 2.10.3 Non-structural protein VP5 21 2.11 Cell death and apoptosis 22 2.11.1 Apoptosis versus necrosis 23 2.11.2 Viruses and viral products induced apoptosis 26

2.12 Nucleoprotein (NP) of NDV strain AF2240 as a molecular carrier 28

3 MATERIALS AND METHODS 3.1 Source of cell and plasmids 30 3.2 Source of chemicals and biochemicals 30 3.3 Construction of recombinant plasmid pTrcHis2- NPfl-VP5 31

3.3.1 Primer design 31 3.3.2 Amplification of VP5 gene by PCR 32

3.3.3 Detection of PCR product 34 3.3.4 Purification of PCR product 34 3.3.5 DNA quantification and purity 35 3.3.6 Extraction of pTrcHis2-NPfl 36 3.3.7 Cloning of VP5 Genes into pTrcHis2-NPfl 37 3.3.7.1 Restriction enzyme digestion 37

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3.3.7.2 Ligation 37 3.3.7.3 Transformation 38 3.3.8 Screening for recombinant plasmid 38 3.3.9 Preparation of glycerol stock 39 3.3.10 Analysis of positive clones 39

3.4 Detection of the expression of recombinant plasmids by Western blot 40

3.5 Solubility test 41 3.6 Subcloning of the NPfl-VP5 and VP5 inserts into mammalian expression vector 43

3.6.1 TOPO TA cloning 47 3.6.2 Analysis of positive clones 48

3.7 Sequencing of the recombinant plasmid 49 3.8 Mammalian cell culture 50 3.8.1 Maintenance of CHO cells 50 3.8.2 Cultivation of CHO cells 51 3.8.3 Preparation of stock culture 52 3.8.4 Transient transfection of mammalian cells 52 3.9 Detection of recombinant protein by Western blot 53 3.10 DNA Laddering 54 3.11 Acridine orange and propidium iodide (AOPI) assay 55 4 RESULTS 4.1 Construction of recombinant plasmid of pTrcHis2-

NPfl -VP5 56 4.1.1 Amplification of VP5 gene by PCR 56 4.1.2 Cloning of VP5 genes into pTrcHis2-NPfl 59

4.2 Analysis of positive clones of pTrcHis2-NPfl-VP5 59 4.3 Detection of the expression of the recombinant protein by Western blot 62

4.4 Solubility test 65 4.5 Subcloning of the NPfl-VP5 and VP5 into pcDNA3.1/V5-His TOPO 67

4.5.1 Amplification of NPfl-VP5 and VP5 gene 67 4.5.2 Analysis of positive clones 67

4.6 Determination of orientation and in-frame location of recombinant plasmids. 75 4.7 Transient transfection of mammalian cells 78 4.8 Detection of recombinant protein by Western blot 80 4.9 DNA Laddering 80 4.10 Acridine orange and propidium iodide (AOPI) assay 84 4.11 Quantification of apoptotic cells and statistical analysis 87 5 DISCUSSION 92 6 CONCLUSSION 103 REFERENCES 104 APPENDICES 122 BIODATA OF THE AUTHOR

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

CLONING AND EXPRESSION OF NUCLEOCAPSID PROTEIN OF NEWCASTLE DISEASE VIRUS FUSED WITH VP5 GENE OF INFECTIOUS

BURSAL DISEASE VIRUS

By

SHAHERNY ZAID

June 2007 Chairman: Datin Paduka Professor Khatijah Mohd. Yusoff, PhD Faculty: Biotechnology and Biomolecular Sciences

Infectious Bursal Disease Virus (IBDV) is a constant threat to the poultry

industry worldwide. This virus encodes a non-structural protein VP5 (17 kDa)

that is capable of inducing apoptosis and may play an important role in

pathogenesis of IBD. The nucleoprotein (NP) of Newcastle Disease Virus

(NDV) has been shown possible to be use as a general carrier. In this study,

the C-terminal end of NP full-length (pTrcHis2) were fused to the full-length of

VP5 of IBDV to study the effect of these construct in cell culture and the

possibility of this recombinant to induce apoptosis. The clones were then

expressed and solubility test were done to determine the level of solubility of

the recombinant proteins by Western blot. The DNA insert of NPfl-VP5 and

VP5 were further subcloned into pCDNA3.1/V5 TOPO TA vector. The

pcDNA3.1-NPfl-VP5 and pcDNA3.1-VP5 constructs were tested for transient

expression in Chinese hamster ovary (CHO) cells. DNA laddering analysis

and Acridine orange propidium iodide (AOPI) was also performed in order to

check for any changes due to apoptosis. Expression of the NPfl-VP5 protein

in E. coli was not detected by SDS-PAGE but a band at the expected size (~

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70-80 kDa) was detected using Western. Only 5% of this protein was soluble.

The PCR products of NPfl-VP5 (1.9 kb) and VP5 (447 bp) were subcloned

into pcDNA3.1 vector. The CHO cells that were used in in vitro expression

were successfully transfected with the plasmid contain the inserts. After 48

hours post-transfection, the cells that were transfected with pcDNA3.1-NPfl-

VP5 and pcDNA3.1-VP5 plasmids exhibited apoptosis. In contrast, no

apoptosis was observed in the cells transfected with pcDNA3.1 vector only.

To confirm that apoptosis had occurred, at 48 hours post-transfection, the

cellular DNA was extracted and analyzed by 2% agarose gel electrophoresis.

A laddering effect indicative of nucleosomal fragmentation, was detected in

the DNA samples obtained from the cells transfected with pcDNA3.1-

NPflVP5 or with pcDNA3.1-VP5 but not with the samples from cells

transfected with pcDNA3.1 vector only. The expression of NPfl-VP5 and VP5

proteins in transfected CHO cells were detected by Western blot analysis

using anti-V5, anti-NDV, anti-IBDV and anti-VP5 antibodies. Further

investigation using AOPI methods also confirm the apoptosis effects.

Statistical analysis showed that the percentage of apoptosis cells was

significantly higher when transfected with pcDNA3.1-NPflVP5 compare with

pcDNA3.1-VP5. As a conclusion, the recombinant protein was successfully

constructed and NP can be used as a carrier, and the fusion of NP to VP5

will induce more apoptosis. However, it just slightly increases its solubility.

Thus, the results showed that recombinant protein NPfl-VP5 and also VP5

alone were able to induce apoptosis in cell culture.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENGKLONAN DAN PENGEKSPRESAN GABUNGAN PROTIN

NUKLEOCAPSID VIRUS PENYAKIT NEWCASTLE DENGAN GEN VP5 VIRUS PENYAKIT BURSA BERJANGKIT

Oleh

SHAHERNY ZAID

June 2007

Pengerusi: Datin Paduka Profesor Khatijah Mohd. Yusoff, PhD Fakulti: Biotechnologi dan Sains Biomolekul Virus penyakit bursa berjangkit (IBDV) merupakan ancaman yang berterusan

kepada industri ternakan ayam seluruh dunia. Virus ini mempunyai protein

yang tidak berstruktur VP5 (17 kDa) yang mampu untuk meransang

apoptosis dan mungkin memainkan peranan penting didalam proses

patogenesis IBD. Nukleoprotein (NP) Virus Penyakit Newcastle (NDV) telah

dikatakan berkemungkinan berguna sebagai pembawa umum. Oleh itu,

didalam kajian ini, hujung terminal-C NP (pTrcHis2) telah digabungkan

dengan VP5 penuh untuk mengkaji kesan penggabungan ini didalam kultur

sel dan kemungkinan gabungan ini untuk meransang apoptosis. Klon-klon ini

telah diekspreskan dan ujian kelarutan telah dilakukan untuk menentukan

tahap kelarutan protein rekombinan dengan Western blot. Seterusnya NPfl-

VP5 dan VP5 telah disubklon ke dalam vector pCDNA3.1/V5 TOPO TA yang

kemudiannya telah di uji untuk pengekspresan ‘transient’ didalam sel

Chinese hamster ovary (CHO). Analisis ‘DNA Laddering’ dan ‘Acridine

orange propidium iodide’ (AOPI) juga telah dijalankan untuk meneliti

sebarang perubahan yang disebabkan oleh apoptosis. Pengekspresan

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protein NPfl-VP5 didalam E. coli tidak dapat dikesan menggunakan SDS-

PAGE tetapi jalur saiz yang dijangka (70 ke 80 kDa) telah dikesan dengan

menggunakan Western blot. Hanya 5% daripada protein larut. Produk PCR

NPfl-VP5 (1.9 kb) dan VP5 (447 kb) telah disubklon ke dalam vector

pcDNA3.1. Selepas 48 jam setelah sel CHO ditransfeksi secara in-vitro, sel-

sel yang ditransfekkan dengan plasmid pcDNA3.1-NPfl-VP5 dan pcDNA3.1-

VP5 menunjukkan apoptosis. Sebaliknya, tiada apoptosis dilihat didalam sel-

sel yang ditransfekkan dengan kawalan. Untuk memastikan yang apoptosis

telah berlaku pada 48 jam selepas transfeksi, DNA selular telah diekstrak

dan dianalisa menggunakan 2% gel agarose elektroforesis. Kesan

“bertangga” menandakan pemecahan nukleosomal telah dikesan didalam

sampel DNA yang didapati dari sel-sel yang ditransfekkan dengan

pcDNA3.1-NPflVP5 atau dengan pcDNA3.1-VP5 tetapi tidak dengan sampel

dari sel-sel yang ditransfekkan dengan kawalan. Pengekspresan protein

NPfl-VP5 dan VP5 didalam sel-sel CHO yang ditransfekkan telah dikesan

menggunakan analisa Western blot menggunakan antibodi anti-V5,anti-

NDV,anti-IBDV dan anti-VP5. Kajian AOPI juga mengesahkan kesan

apoptosis. Analisis statistik telah menunjukkan bahawa peratus sel apoptosis

secara signifikan adalah lebih tinggi bila ditransfekkan dengan pcDNA3.1-

NPfl-VP5 berbanding pcDNA3.1-VP5. Sebagai kesimpulan, protein

gabungan telah berjaya dibentuk dan diekspreskan, dan gabungan NP dan

VP5 akan lebih merangsang apoptosis. NP boleh digunakan sebagai

pembawa walaupun hanya meningkatkan sedikit kelarutannya. Keputusan

juga menunjukkan yang protein recombinan NPfl-VP5 dan juga VP5 mampu

untuk meransang apoptosis didalam kultur sel.

ACKNOWLEDGEMENTS

I would like to take this opportunity to thank all people who have helped me

throughout the completion of my Master’s thesis. Without them, this project

would not have been possible.

First of all, I owe my indebtedness to my wonderful supervisor, Prof. Datin Dr.

Khatijah Mohd. Yusoff for introducing me to this field and also for all the

personal attention, excellent advise, guidance, encouragement and support

throughout my research. I also would like to thank my co-supervisors, Assoc.

Prof. Dr. Abdul Rahman Omar and Dr. Muhajir Hamid for their invaluable

comments and suggestions. I also would like to extend my sincere thanks to

my employer, Universiti Pendidikan Sultan Idris (UPSI) and JPA for their

sponsorship for my Master’s programme.

I also would like to acknowledge with sincere gratitude to Dr. Majid Eshagi for

guiding me in tissue culture work. Special thanks to Wan Key Fei for

providing me the cells for my research and also Prof. Manaf Ali and his

students in guiding me in the usage of the fluorescence microscopy.

I am grateful to my laboratory mates, especially to Fida, Sue, TC, Andrew,

Wawa, Taz, Kak Tan, Lalita, Kah Fai, Sing King, Firoozeh, Eddie, Mok, Max,

Yeng Peng, Swee Tin and Chew Ling for assisting me in various ways in my

laboratory work and for giving me motivation. Appreciation also goes to

Nazrien and Tajul for guiding me in AOPI.

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I extend my deepest and warmest thank to my beloved, Zul and my family for

being so helpful, understanding and for having patience in me. Not

forgetting, thanks to all my friends who were always being there for me

during the hard times of my research.

Finally, above of all, I thank God for His blessing on me….

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I certify that an Examination Committee has met on 27th June 2007 to conduct the final examination of Shaherny binti Zaid on her Master of Science thesis entitled “Cloning and Expression of Nucleocapsid Protein (NP) of Newcastle Disease Virus Fused With VP5 Gene of Infectious Bursal Disease Virus” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the degree of Master of Science. Members of the Examination Committee were as follows: Raha Abdul Rahim, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Abdul Manaf Ali, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Mohd. Hair Bejo, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Rahmah Mohamed, PhD Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (External Examiner)

________________________________ HASANAH MOHD. GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Khatijah Mohd Yusoff, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Abdul Rahman Omar, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Muhajir Hamid, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) AINI IDERIS, PhD

Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 22 January 2008

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

________________

SHAHERNY ZAID

Date:

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LIST OF TABLES

Table Page 2.1 Taxonomic structure of the family Birnaviridae. 7

2.2 Differential features and significance of apoptosis and necrosis.

25

2.3 Viruses and viral proteins implicated in inducing apoptosis.

27

3.1 Construction of recombinant plasmid pTrcHis2-NPfl-VP5.

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3.2 Oligonucleotide primers used in amplification of VP5 gene.

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3.3 Oligonucleotide primers used in amplification of NPflVP5 and VP5 gene.

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3.4 Construction of recombinant plasmids pcDNA3.1-NPfl-VP5 and pcDNA3.1-VP5.

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LIST OF FIGURES Figure Page

2.1 Schematic representation of the genomic organization of IBDV.

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3.1 Positions of the oligonucleotides used in amplifying the specific VP5 gene indicated on the complete VP5 gene.

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3.2 Positions of the oligonucleotides used in amplifying the specific NPfl-VP5 and VP5.

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4.1 Agarose gel electrophoresis of amplified VP5 gene (447bp) from pCR2.1-VP5.

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4.2 PCR screening of putative transformants containing the recombinant plasmid of pTrcHis2-VP5.

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4.3 Restriction endonuclease analysis of putative recombinant plasmid pTrcHis2-NPf-lVP5.

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4.4a Western blots analysis of recombinant NPfl-VP5 protein expressed in recombinant E.coli containing the plasmid pTrcHis2-NPfl-VP5. The proteins were analyzed on 12% SDS-PAGE and Western blot against anti-myc.

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4.4b Western blots of recombinant NPfl-VP5 protein expressed in recombinant E.coli containing the plasmid pTrcHis2-NPfl-VP5. The proteins were analyzed on 12% SDS-PAGE and Western blot against anti-NDV and anti IBDV.

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4.5 Western blots of soluble test of NPfl-VP5 protein expressed in recombinant E.coli containing pTrcHis2-NPfl-VP5. The proteins were analyzed on 12% SDS-PAGE and Western blot against anti-NDV.

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4.6 Agarose gel electrophoresis of amplified NPfl-VP5 (1.9 kb) from recombinant pTrcHis2-NPfl-VP5 plasmid.

68

4.7 Amplification of VP5 (447 bp) from pCR2.1-VP5 plasmid.

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4.8 PCR screening of recombinant plasmid of pcDNA3.1-NPfl-VP5.

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4.9 PCR screening of recombinant plasmid of pcDNA3.1-VP5.

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4.10 Restriction endonuclease analysis of pcDNA3.1-NPfl-VP5 (BamHI and XhoI) of the extracted plasmid of pcDNA3.1-NPfl-VP5.

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4.11 Restriction endonuclease analysis of pcDNA3.1-VP5 (BamHI and XhoI) of the extracted plasmid of pcDNA3.1-VP5.

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4.12 Chromatogram of the sequencing result of the recombinant plasmid pTrcHis2-NPfl-VP5.

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4.13 Chromatograms of the sequencing results of the recombinant plasmids of pcDNA3.1-NPfl-VP5 and pcDNA3.1-VP5.

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4.14 The effect of recombinant NPfl-VP5 and VP5 protein on CHO cell at 48 h post transfection.

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4.15a Detection of the recombinant NPfl-VP5 and VP5 protein expression in CHO cells by imunobloting against anti-V5 and anti-NDV.

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4.15b Detection of the recombinant NPfl-VP5 and VP5 protein expression in transfected CHO cells by imunobloting against anti-IBDV and anti VP5.

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4.16 Detection of the cellular DNA fragmentation induced by transient expression of recombinant NPfl-VP5 and VP5 protein in CHO cells.

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4.17 Induction of apoptosis in CHO cells transfected with the expression vectors pCDNA3.1-NPfl-VP5.

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4.18 Induction of apoptosis in CHO cells transfected with the expression vectors pCDNA3.1-VP5.

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4.19 Histogram of the percentages of mode of cell death at 16 hours post- transfection with the indicated recombinant plasmids.

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4.20 Histogram of the percentages of mode of cell death at 24 hours post- transfection with the indicated recombinant plasmids.

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4.21 Histogram of the percentages of mode of cell death at 48 hours post- transfection with the indicated recombinant plasmids.

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LIST OF ABBREVIATIONS amp ampicillin

AOPI Acridine orange propidium iodide

ATP Adenosine triphosphate

β-Gal β-Galactosidase

bp base pair

C Carboxy

cDNA copy DNA

CMV Cytomegalovirus

CO2 Corbon dioxide

DMSO Dimetyl sulfoxide

DNA Deoxyribonucleic Acid

dsDNA double-stranded DNA

dsRNA double-stranded RNA

EDTA Ethylene diamenetetraacetate

EM Electron microscopy

FBS Foetal bovine serum

HCI Hidrocloric acid

IBD Infectious Bursal Disease

IBDV Infectious Bursal Disease Virus

kb kilobase

kDa kilo Dalton

Mab Monoclonal antibody

mRNA messenger RNA

NaCI Sodium chloride

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NDV Newcastle Disease Virus

nt nucleotide

OD Optical density

OIE Office International des Epizooties

ORF Open reading frame

PBS Phosphate-buffered saline

PCR Polymerase chain reaction

p.i. post-infection

RdRp RNA-dependent RNA polymerase

RNA Riboucleic acid

RNAse Ribonuclease

rpm Rotation per minute

SDS-PAGE Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis

ssDNA single-stranded DNA

ssRNA single-stranded RNA

UPM Universiti Putra Malaysia

VLP Virus-like Particle

VP Viral protein

VPg genome-linked viral protein

vvIBDV very virulent IBDV

CHAPTER 1

INTRODUCTION

Infectious bursal disease virus (IBDV) is a major important pathogen to the

poultry industry worldwide, causing a highly contagious immunosuppressive

disease in young chickens by destroying the lymphoid cells in the bursa of

Fabricius (BF) (Mundt et al.,1995). In addition, the immunosuppression

increases the susceptibility of these chickens to other infections and

interferes with vaccination against other diseases (Saif,1991).

IBDV is a small, icosahedral and non-enveloped virus which belongs to the

genus Avibirnavirus of the family Birnaviridae (Murphy et al., 1995). There

are two serotypes of IBDV; serotype 1 strains are pathogenic to chickens

whereas serotype 2 strains are not (Ismail et al., 1988). The virus contains a

bisegmented double-stranded RNA genome, namely segments A and B.

The larger Segment A encodes three structural virion proteins, VP2, VP3 and

VP4, and a nonstructural protein, VP5 (Mundt et al., 1997). The smaller

segment B, encodes VP1, the viral RNA polymerase (Muller & Nitschke,

1987). VP2 has been identified as the host protective antigen as it contains

the antigenic regions responsible for the induction of neutralizing antibodies

(Fahey et al., 1989). VP3 is considered to be a group specific antigen as it is

only recognized by non-neutralizing antibodies, some of which cross-react

with serotypes 1 and 2 (Oppling et al., 1991). VP4 is a viral protease involved

in the processing of the polyprotein (Azad et al., 1987). It has been

demonstrated that VP5 is neither required for the viral replication in vitro

(Mundt et al., 1997) nor in vivo but plays an important role in pathogenesis as

proven by the generation of a VP5-defective virus which had lost ability to

cause bursal lesions (Yao et al., 1998).

The use of a reverse genetics system has allowed the generation of a VP5

knockout mutant. Work with this VP5 deletion mutant has shown that VP5 is

dispensable for virus replication in vitro (Mundt et al., 1997). Interestingly,

this mutant showed a highly attenuated phenotype which did not cause

bursal lesions after experimental infection of susceptible chickens,

demonstrating that VP5 plays a key role in IBDV pathogenesis and

dissemination (Yao et al.,1998). Consequently, VP5 has attracted attention

as a potential target in the development of strategies to control IBDV. The

study by Yao et al. (1998) indicated that the absence of VP5 protein

expression in the mutant virus will not affect the humoral immune response to

IBDV in the natural host. Therefore, it may be feasible to develop a novel,

live-attenuated vaccines for IBDV that are non-pathogenic to chickens.

Although several improvements in the control of the diseases by vaccination

have been obtained, the results remain unsatisfactory. Despite strong

interest in VP5, the Birnavirus life cycle has not been characterized in detail.

Very little is known on the virus entry, genome transcription and replication,

morphogenesis, and release of the viral progeny.

Studies have shown that immunosuppresssion induced by IBDV is caused, at

least in part, by apoptosis or programmed cell death (Tanimura & Sharma,

2

1998). Recently, it has been shown that an IBDV infection induces apoptosis

in chicken embryo and tissue culture cells (Yao and Vakharia, 2001). VP5

alone is capable of inducing apoptosis in cell culture. Transfection of chicken

B-lymphocyte cell line (RP9) and chicken embryo fibroblast cells with a

plasmid DNA containing the VP5 gene under the control of the intermediate-

early promoter enhancer region of human cytomegalovirus, induced

apoptosis in both cell lines. Apoptotic changes such as chromatin

condensation, DNA fragmentation, and the appearance of apoptotic nuclear

bodies were observed in cell cultures 48-h post-transfection (Yao and

Vakharia, 2001). In addition, induction of VP5 expression also resulted in the

alteration of cell morphology, the disruption of the plasma membrane, and a

drastic reduction of cell viability. These observations indicate that the VP5 is

highly cytotoxic and is involved in the release of the viral progeny from cells,

an important role in viral pathogenesis. (Lombardo et al., 2000; Yao and

Vakharia., 2001).

The nucleoprotein (NP) of Newcastle disease virus (NDV) has recently been

expressed in Escherichia coli where it assembles into ring-like particles. It

has been suggested that the NP has the potential to be developed as a

carrier for chimeric antigens (Kho et al, 2001b). In addition, it can be

expressed in abundance as a highly soluble protein. This protein is stable

and has its C-terminus exposed making it ideal for carrying any antigen or

multiple antigens (Rabu et al, 2002). Therefore, it would be interesting to

know whether the fusion of NP with VP5 might increase the efficacy of the

latter in inducing apoptosis compared to VP5 alone, since fusion of NP with

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HN and F proteins of NDV induced significant levels of antibodies against

NDV in chickens (Rabu et al., 2002). With the use of NP as a carrier, NP-VP5

might be expressed in soluble and stable form due to the ability of NP, which

can act as a carrier for other peptide (Rabu et al., 2001).

Despite a number of studies on IBDV, very little is known about the

expression of the VP5 gene of IBDV or on a recombinant VP5. In this study,

the full length of VP5 were fused to the C-terminal of NP to study the role and

effect of chimera NP-VP5 and a possibility of this chimera could induce

apoptosis. Hence, the purpose of fusing NP of NDV with VP5 of IBDV in this

study was to increase the efficacy of VP5 in inducing apoptosis in normal

mammalian cells.

Therefore the objectives of this study are:

1. to construct recombinant DNA molecules carrying NP of NDV and VP5

gene of IBDV;

2. to express the chimeric protein in a mammalian expression system; and

to study its effect on apoptosis in vitro.

CHAPTER 2

LITERATURE REVIEW

2.1 IBDV and the disease

Infectious bursal disease (IBD) is a highly contagious, immunosuppressive

disease of young chickens caused by the infectious bursal disease virus

(IBDV). The disease is characterized by destruction of the lymphoid cells in

the bursa of Fabricius (BF), which leads to severe B-cell suppression (Becht,

1980; Kibenge et al., 1988) resulting in susceptibility to other infections and

interferences with vaccination programmes (McFerran et al., 1980; Snyder et

al., 1988). The infection depends on the dose and virulence of the strain, the

age and breed of animals, and the presence or absence of passive immunity

(Tacken et al., 2000). Nevertheless, the disease has no direct impact on

public health as there is no evidence of transmission of IBDV to other non-

avian species including human.

2.2 History and epidemiology

The disease was discovered in 1962 in Gumboro, Delaware, USA (Cosgrove,

1962) but had been present since 1957 (Cover, 1960). Later outbreaks were

subsequently referred to as ‘Gumboro Disease’ after the geographic location.

Originally the condition was named ‘nephritis-nephrosis syndrome of chicken’

because of prominent kidney lesions. After it became evident that an

5