UNIVERSITI PUTRA MALAYSIA

CLONING AND EXPRESSION OF THE HAEMAGGLUTININ-NEURAMINIDASE (HN) GENE FROM NEWCASTLE DISEASE VIRUS

(NDV) STRAIN AF2240 IN BACULOVIRUS (AcMNPV)

ALAN ONG HAN KIAT

FSAS 1999 26

CLONING AND EXPRESSION OF THE HAEMAGGLUTININ­NEURAMINIDASE (HN) GENE FROM NEWCASTLE DISEASE VIRUS

(NDV) STRAIN AF2240 IN BACULOVIRUS (AcMNPV)

By

Alan Ong Han Kiat

Thesis Submitted in Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of

Science and Environmental Studies U niversiti Putra Malaysia

September 1999

ACKNOWLEDGEMENTS

It gives me great pleasure to express my deepest appreciation and gratitude to

Assoc Prof Dr Khatijah Mohd Yusoff, Assoc Prof Dr Abdul Manaf Ali and Dr

Abdul Rahman Omar for theIr advice, guidance and support m supervIsing this

project

With all its challengers and complexity, this Ph D theSIS would not have

come true had It not been for the love, understanding and encouragement of my

famIly, love one and friends

My gratitude also goes out to a number of kind and helpful people who have

played a major role in the course of the project especially Assoc Prof Kazuaki

Takehara, Assoc Prof Dr Abdullah Sipat and Dr Tan Wen Siang for their advice,

Mr Poh Yang Ming for conducting part of the EM and optimisation studies and not

forgetting Prof Dr AmI Idens, Dr K Ganapathy and the Staff of Vaccine lab for

theIr suppon in the field trial experiment

It has also been a great expenence and a joy workmg with my colleagues m

Lab 143, 202 of the Depanment of Biochemistry and Microbiology as well as the

Ammal Tissue Culture Lab in the Department of Biotechnology I am also very

grateful for the technical assistance rendered by staffs from the Department of

II

Biochemistry and Microbiology, Virology Lab and Electron Microscopy Unit in the

F acuity of Veterinary Medicine.

Mr. Ng Chong Sin, Mr. Julian Lim from Bio-Diagnostic, Madam Kee Ng

form Life Technologies, Mr. Law from BioSyntax and Mr. Yeo Wee Kiat from

Boehringer Mannheim have also contributed significantly in terms of technical

advice and support.

Finally, I would like to thank God for His spiritual guidance and for blessing

me with all those wonderful people mention above as well as many more who have

contributed one way or another.

This study was supported by the Malaysian Ministry of Science, Technology

and Environment IRP A Grant no. 01-02-04-0107.

III

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS LIST OF TABLES

11 IX X Xl

LIST OF FIGURES LIST OF PLATES LIST OF ABBREV ATIONS ABSTRACT

Xlll XV

XVll ABSTRAK

CHAPTER

I

II

INTRODUCTION

LITERATURE REVIEW Newcastle Disease Virus

Taxonomic Classification History and Distribution ofNDV Newcastle Disease Virus and the Malaysia Scenario Disease Control of NDV and the Challenges it Poses in Malaysia NDV Genome, Encoded Protein and Virion Structure

NDV Stram AF2240 Pathogenic, Morphology and Molecular Characteristics The Molecular Characteristics of the Haemagglutinin-Neuraminidase (HN) Glycoprotem

Page

1

5 5 5 6 6

7

9 1 1

11

ofNDV Strain AF2240 13 The Sigruficance of the HN Glycoprotein 1 6

The Role of the HN Surface Glycoprotein in the Viral Llfe Cycle (InfectlOn) 1 6 Role of the HN Surface Glycoprotein in the Host Immune Response 1 7 Immunological Implications of the HN Glycoprotein 19 The Immunological Importance of Glycosylation in the HN Protein ofNDV 2 1

lV

III

The Biology of B aculovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Baculovirus Classification . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . Baculovirus Virion and Genome Structure . . . . . . .... . . Effects of Serial Passage of Virus . . . . . . . . . . . . . . . . . . . . . . .

Baculovirus Expression Vector System ( BEVS) . . . . . . . . . . . . . . Principles of BEVS . . . . . . . .. . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . Advantages of The BEVS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Baculovirus Transfer Vectors . . . . . . . . . . . . . . . . . , . . . . . . . . . Insect Cell Spodoptera jrugiperda (Sj9) Culture . . . . . . Characteristics of Insect Cell Lines . . . . .. . . . . . . . . . . . . . . . Insect Cell Culture Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maintenance of Sj9 Insect Cells . .. . . . . . . . . . . . . . . . . . . . . . Co-Transfection of Baculovirus DNA and the Transfer Vector into Insect Cells . . . .. . . . . . . . . . . . .. . . . .

The Recombinant Subunit Vaccine For the Potential Control of Newcastle Disease Virus .. . . . ... . . . . . . . . . . . . . ... . . . .

Current Vaccines . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . Recombinant Subunit Vaccine . . . . . . . . . . . . . . . . . . . . . . . . .

MATERIALS AND NfETHODS . . . . . . .. . . . . . . . . . . . . ...... . .. . . .

23 23 24 25 28 28 29 31 32 ..,.., JJ 33 35

35

37 37 39

41 General Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Viruses . ... _ . . . .. . . . . . . .. _ . . . .... _ _ _ . _ _ . . ..... . _ _ _ _ .. . _ _ . . . . . 41 Bacteria. . . . . . . . . . . . . . . . . . ... . . . .. . . .. . . . . . . . .. . . . . . . . . . .. . . 42 Cell lines . . . . . . '" .... , .... '" '" ... ... ... ... ... ... ... ... ... 42

Virus Cultivation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Newcastle Disease Virus ( NDV) . . . . . . . . . . . . . . . . . ... .... 43 Wild type (wt) Baculovirus (AcMNPV). . . . . . . . . . . . . . . 43

Nucleic Acid Extraction . . . . . , '" '" ...... '" '" ... ... ... ... ... ... 44 Viral RNA Extraction of NDV . . . . . . . . . . . . '" ... '" .... 44 Viral DNA Extraction from Infected Sj9 Insect Cells . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . .. . 44

Concentration and Purity of Extracted Nucleic Acids. . . . . ... 46 Polymerase Chain Reaction ( PCR) Procedures . . . . . . . . . . . . .. . 47

Primers Used.. . . ...... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 47 RT-PCR Amplification......... . .. . . . ... . . . ............. 48

Cloning Procedures ... .. . ......... . .. ............ ................. 51 Purification of RT- PC R Products . . .... . ... . . . .. . . .. _ _ 51 Ligation of RT-PCR Products and The Transfer Vector ( pCR Bac 4.8 kb) . .. . ... . .. . . . . . . . . . . . . . . . . ... . . 52 Transformation of The Recombinant Transfer Vector Into Competent E. coli Cells . . . .. . ... ........... 53 TO PO TA Cloning for Sequencing . . . . . . . . . . . . . . , . " " 53 Subcloning of the Insert from TO P O Vector pCR 2.1 into Baculovirus Transfer Vector pCR Bac 4.8 56

v

Screening and Verification of Putative Recombinant

Clones 58 Small Scale Plasmid Extraction (Plasmid Miniprep) 58 Restriction Enzyme Analysis 59 Polymerase Cham Reaction (PCR) 60 DNA Sequencing 60

Co-Transfection of the Recombinant Transfer Vector and the Lmearlsed Baculoviral (Ac:MNPV) DNA into Sj9 Insect Cells 62

Sj9 Cells 62 TransfectIOn Mixture 62 Co-transfection into Sj9 Cells 63 Harvesting 63

IdentIficatIon and Verification 64 Viral Plaque Assay 64 PCR Verification 65 Partial Sequencing at the End Terminals of the RecombInant Viral DNA 66

Propagation of Large-Scale High Titer (P3) Recombinant Virus (recHNAF2240) Stock 66

SpInner Flask Culture 66 DetermmatIOn ofP3 Virus Titer 67

Electron Microscopy of Sj9 Cells and Virus 67 NegatIve Staining of Viral Samples 67 TEM of Viral Infected Sj9 C ells 68

Serological Test 69 Processing of Viral Samples form Infected Sj9 Cells 69 HaemagglutInlfi Activity (HA) 70 HemadsorptIOn ActIVIty 7 1 Neuraminidase ActIVIty (NA) 7 1 Haemagglutmation Inhibition (ill) 72 HemadsorptIOn InhibitiOn 72 Indirect Immunofluorescence (IIF') 72

Protein Analysis 73 Sodium Dodecyl Sulphate- PolyacrylamIde Gel ElectrophoresIs (SDS-PAGE) 73 Western BlottIng 75 Immuno Detection of Transferred Proteins on PVDF Membranes 76

Time Course Study for the ProductIOn of Recombinant HN Protein 77 Small Scale OptImIsation Study on the Recombinant Protein Expression 78 Large Scale Production of Recombinant HN Protein 79

VI

IV

Preliminary Field Trial Immunisation Study of the Recombinant HN Subunit Vaccine.............................. 79

Preparation and Quantification of the Subunit Vaccine................................................ ..... 80 Layout of the Vaccination Field Trial... . . . . . . . . . . . . ... 82 Collection of Sera from Vaccinated and Unvaccinated Chickens.................................. 84 Challenge Procedure... ......... .......................... 86

Analysis of the Immune Response from Vaccinated Birds 86 HI Test...................................................... 86 Enzyme Link Immunoabsorbent Assay (ELISA)..... 87

RESULTS At'ID DISCUSSION ................ " . ............ . 89 Reverse Transcriptase - Polemerase Chain Reaction CRT-PCR) of Viral R.,.1\JA......................... ..................... 89 Optimised One-Step RT-PCR for Cloning... ...... ....... .... 91 Cloning of the HN Gene into a Baculovirus Transfer Vector............................................................... 94 Screening and Verification of Putative Recombinant Clones............................................................... 95

Plasmid Extraction and Restriction Enzyme 95 Analysis ................................................... . PCR Verification .................................... , ... , 97 Partial End Terminal Sequencing of the Putative Recombinant Plasmid.................................... 100 TOPO Cloning for Sequencing and Sub-Cloning.... 102

Common Morphology of Sj9 Insect Cell Culture.............. 104 Co-Transfection into Sj9 Insect Cells........................... 104

Visual Confirmation ... '" ... ... ... ... ... ... ... ... ... ... .. 104 Identification and Verification .... , .................... , 105

Large Scale Propagation of Recombinant Virus ......... '" ... 114 Electron Microscopy of the Virus and Sj9 Cells.......... .... 114

EM of Newcastle Disease Virus........................ 114 EM of the Wild-Type (wt) and Recombinant (recHNAF2240) Baculovirus........................... 114

Expression of the HN Recombinant Baculovirus in Sj9 Cells ........................... . ... . . ... _ ......................... _ . . 124

Serological Test.................................... ... .... 124 Inhibition Assays.................. .................. ...... 129 Protein Analysis with SDS-PAGE and Western Blotting................................................... 132 Time Course Study for the Production of Recombinant HN Protein.................. .............. 136

Small Scale Optimisation Study on the Recombinant Protein Expression........................... ...... ............... 142

vii

V

Large Scale Production ofRecomhinant HN Protein 148 Preliminary Field Trial Immunisation Study 1 5 1

Quantification of Subunit Vaccine 1 5 1 Immunisation Trials 1 5 1

Analysis of the Immune Response from Experiment 1 and Experiment 2 1 54

HI Test 1 54 Challenge Experiment 1 55 ELISA 1 6 1 Western Blot Analysis 1 65

Overall Discussion 1 7 1

CONCLUSION 1 76

REFERENCES 1 78 APPENDIX

A 2 12 B- 1 2 1 3 B-2 2 1 5

VITA 2 1 9

VIIl

Table

2

" .) 4 5

6

7

8

9

1 0

1 1

12

1 3

LIST OF TABLES

The Concentration and Punty of the Extracted Nucleic Acids List of Primers Used for RT -PCR PCR .A.nalysis and Sequencing Worksheet for the Vaccination of Experiment 1 Worksheet for Immunization of Chickens in Experiment 2 Names of Cloning Vectors and Recombinant Plasmlds Generated from the Various Clomng Methods Quantification of Viral and Total Protein Sample from WT and Recombmant Subunit Vaccine Preparation for Experiment 1 Quantification of Viral and Total Protein Sample from WT and Recombmant Subunit Vaccine Preparation for Experiment 2 Mean ill (Log2) of Serum Samples Taken from Experiment 1 ill Titers (Log2) of Serum Samples form Chickens Vaccinated with the Recombinant Subunit Vaccine (RecHN AF2240) taken from Experiment 2 Mean ELISA Absorbance Values with NDV Precoated Plates (KPL) of Seruma from the 1 st Experiment Mean ELISA Absorbance Values with NDV Precoated Plates (KPL) of Seruma from the 2nd Experiment ELISA Absorbance Values with NDV Strain AF2240* Coated Plate of Seruma Taken from Each Group ELISA Absorbance Values with the Recombinant Baculovirus (recHNAF2240)* Coated Plate of Serum3 Taken from Each Group

IX

Page

46

48 84 88

107

158

159

162

1 63

168

169

172

1 73

LIST OF FIGURES

Figure Page

1 Comparison of Product lOn, Consumption and Prices of Local Meat Source 2

2 Schematic Diagram of a Paramyxovmdae 1 0 .., Schematlc DIagram of a) Budded Virus (BV) and b) -'

Occlusion ViruS (OV) of Ac:NfNPV 26 4 The Infection Cycle of the Baculovlrus BV and OV In

Insect Cells 27 5 The pCR Bac 4 8 Baculovlrus Transfer Vector with the T A

Cloning and the MultIple ClonIng Sites 53 6 Schematic Diagram of the ClonIng Strategy. 54 7 The pCR 2 1 TOPO T A Clomng Vector ContaInmg the

Topoisomerase (Tn and the Related Multip le Cloning Sites 59

8 Schematic DIagram Showmg the HmdIII Site of the C loned HN Gene Amplified from Viral RNA and the Transfer Vector 102

9 Homologous RecombInation of the Linearized Bac-N-Blue Baculovirus DNA and the Baculovirus Transfer Vector 1 1 8

10 Growth o f Sj9 Insect Cells in TNM-FH Medium with 5% F BS 1 49

1 1 ExpresslOn of the Recombinant Protein in terms ofHAU at Two Different Time of InfectlOn Corresponding to Various mOl Values 1 50

x

Plate

1 2

3

4 5

6

7

8

9

1 0 1 1 1 2

LIST OF PLATES

Administration of Vaccines through the Intranasal Route Administration of Subunit Vaccines through the Intra Muscular Route Amplified RT-PCR Products Using Various Primer Combinations Detected on a 0 8% Agarose Gel P lasmid that have been Digested with BamID and EcoRI Restriction Enzyme Analysis of the Putative Recombinant Plasmid with HmdUI End Terminal Sequencing on Positive Clones from the Directional Cloning Morphology of Transfected Sj9 Insect Cells at Various Time Point Plaque Assay Showing Putative Blue Recombinant Viral Plaques Viral DNA Extraction from Infected Sj9 Cell Cultures Used for the PCR VerificatIOn A Negative Staining Electron Microscope Picture ofNDV Negative Staining of Recombinant and wt Baculovirus EM of Cross Section from Infected and Uninfected Sj9 Cells

1 3 EM of Cross Section from Cells Infected with wt and

14

1 5

16 17 1 8 1 9 20

2 1

22

Recombinant Virus at 2 d p i EM of Cross SectIon from Cells Infected with wt Bacuiovirus at 4 d p i Showing the Presence of the Polyhedrin Sturcture (P) EM of Recombinant Viral Infected Cells Showing Circular-Like Capsids Haemagglutinin Activity (HA) Test wIth 1 % ChIcken RBC NA Test on the Infected Cell Samples HemadsorptIOn Assay wIth Chicken Red Blood Cells InhIbItIOn Assays Immunofluorescence (IIF) of �1 and Recombmant Infected Cells A 12% SDS-PAGE Gel Stamed with Coomassle Blue Comparing Banding Patterns of Infected Cell Lysates Western Blot Analysis of Various Virus Infected Cell Lysates Using NDV Polyclonal Anti-sera

XI

Page

8 1

81

92 96

99

1 0 1

1 06

1 07

1 09 1 16 1 1 7

1 19

1 20

1 2 1

1 22 1 25 1 26 1 27 1 30

1 3 1

1 3 3

1 34

23

24

25

26 27

An Autoradiogram of esS] Methionine-Labeled Samples Showing the Time Course of Vral Protein Synthesis in the Recombinant (R) and Wild Type AcMNPV (WT) Baculovirus Infected Cells . . . . . . . . . . . , . . , . . , . . , . . . . . . . . . . . . . . . . .

An Autoradiogram Showing CSS] Methionine-Labeled Samples of Infected Cell Lysates of Recombinant Baculovirus (recHNAF2240) and Wild Ttype Baculovirus at 48 h .p . i . with and without the Treatment of Tunicamycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Clinical Signs of Viral Infection Observed on Chickens Challenge with the VVNDV Strain AF2240 . . . . . . . . . . , . . . . . . . .

Obvious Signs ofNDV Strain AF2240 Viral Infection . . . . . .

Western B lot Analysis Using the Anti-sera from Recombinant Vaccinated Birds of the 2nd Experiment on the Various Virus Infected Cell Lysates . . . . . . . . . . . . . . . . . . . . . .

XII

1 3 7

1 3 8

1 59 1 60

1 68

AcMNPV

BEVS

BV

cDNA

DNA

dNTP

ddNTP

EDTA

EM

F

HA

HN

kb

kDa

MAb

Mr

NA

NDV

O V

LIST OF ABBREVIA nONS

Autographa californica multiple nuclear polyhedrosis

Virus

Baculovirus Expression Vector System

Budded virus

complementary deoxyribonucleic acid

deoxyribonucleic acid

deoxynucieotide triphosphate

dideoxynucleotide triphosphates

Ethylenediaminetetraacetic acid disodium salt

Electron Microscope

fusion protein

haemagglutinin activity

haem agglutinin-neuraminidase

kilobase

kilodalton

monoclonal antibody

molecular weight

neuraminidase activity

Newcastle disease virus

occlusion virus

XllI

ORF

PBS

PCR

RBC

RNA

RT-PCR

SDS-PAGE

Sj9

Tag

TBE

VVNDV

wt

open reading frame

phosphate buffer saline

polymerase chain reaction

red blood cells

ribonucleic acid

reverse transcriptase-polymerase chain reaction

sodium dodecyl sulphate-polyacrylamide gel

electrophoresis

Spodoptera jrugrperda

Thermus aquaticus

Tris-boric-EDT A buffer

viscerotropic-velogenic NDV

wild type

XIV

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Doctor of Philosophy

CLONING AND EXPRESSION OF THE HAEMAGGLUTININ­NEURAMINIDASE (HN) GENE FROM NEWCASTLE DISEASE VIRUS

(NDV) STRAIN AF2240 IN BACULOVIRUS (AcMNPV)

By

Alan Ong Han Kiat

September 1999

Chairman : Assoc. Prof. Kbatijah Mohd. Yusoff, Ph.D.

Faculty : Science and Environmental Studies

Newcastle disease virus strain AF2240 is a major threat to the poultry

industry as it causes 100% mortality to susceptible flocks The haemagglutinin-

neuraminidase (HN) gene encodes for the fIN surface glycoprotein which is known

for virus attachment and contains immunogenic properties Therefore, the HN gene

was cloned into a Baculovirus Expression Vector system (BEVS) for the

development of a subunit vaccine against NDV as well as to study its expression in

isolation from the other NDV structural genes

The approach taken involved the amplification (RT-PCR) of the I 8 kb HN

gene, from NDV strain AF2240 genomic RNA and cloning it into a BEVS The

xv

recombinant baculovirus protein RecHNAF2240 expressed in Sj9 cells was shown to

be positive for the haemagglutinin test (RA), neuraminidase test (NA), indirect

immunofluorescence (IIF) as well as in SDS-PAGE Western blot analysis indicated

the distinct - 63 kDa and - 75 kDa protein bands as HN specific which corresponded

to the unglycosylated and glycosylated HN glycoproteins respectively This

observation was confirmed by a time course study using pulsed-labeled esS]

methionine of the HN glycoprotein in the recombinant virus infected cells with

tunicamycin The recombinant protein was expressed not only on the surfaces of the

infected cells and the viral coat protein but also appears to be biologically active and

functional The physical nature of the viruses was also studied using electron

microscopy (EM) and it indicated various physical differences

Optimisation study on the recombinant protein RecHNAF2240 production

showed that a late log phase infection of the recombinant virus (recHNAF2240) at an

m 0 i of 1 was the most appropriate Based on HI, ELISA and western blot analysis

the recombinant subunit vaccine was able to elicit a protective immune response

The route of vaccination and a second dose were crucial to illicit an immune

response from the chickens However, thiS protective feature of the recombinant

subunit vaccine remains mconclusIve and more work should be carried out to bring

about the fullest potential of this recombinant subunit vaccine

XVI

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia untuk memenuhi keperluan ijazah Doktor Falsafah

PENGKLONAN DAN PENGEKSPRESAN GEN HEAMGGLUTININ­NEURAMINIDASE (HN) DARIPADA VIRUS PENY AKIT NEWCASTLE

(NDV) STRAIN AF2240 KE DALAM BACULOVIRUS (AcMNPV)

Oleh

Alan Ong Han Kiat

September 1999

Pengerusi : Prof. Madya Khatijah Mohd. YusofT, Ph.D.

Faculti : Sains and P�ngajian Alam Sekitar

Virus penyakit Newcastle strain AF2240 merupakan penyakit virus utama

dalam industri penternakan ayam kerana boleh menyebabkan 100% kematian ayam

Gen HN yang mengkodkan glikoprotein permukaan HN memainkan peranan dalam

perlekatan virus dan juga mempunyai nilai-nilai keimunan Oleh itu, gene HN telah

diklonkan dalam Sistem pengekspresan vektor Baculovirus (BEVS) untuk

penghasilan vaksin subunit terhadap NDV dan juga untuk mengkaji pengekspresan

gen HN berasingan dengan gen-gen NDV yang lain

Pendekatan yang digunakan adalah dengan melibatkan gen HN (1 8 kb) yang

telah diamplifikasikan daripada jujukan RNA (RT-peR) dan diklonkan ke dalam

XVll

BEVS. Protein rekombinan baculovirus RecHNAF2240 yang diekpreskan dalam sel

Sj9 menunjukkan keputusan yang positif untuk ujian hemaglutinin (HA),

neuraminidase (NA), imunopendarfluoran (IIF) serta SDS-P AGE. Analisis "western

blot" pula menunjukkan jalur protein - 63 kDa dan � 75 kDa sebagai specifik kepada

protein HN yang bersamaan dengan glikoprotein HN yang masing-masing tidak

mengalami glikosilasi dan yang mengalami glikosilasi. Keputusan tersebut juga

telah didapati dengan menggunakan protein rekombinan yang dilabelkan dengan

CSS] methionin dengan kehadiran antibiotic tunikamisin. Penemuan sedemikian

membuktikan bahawa protein rekombinan diekspr�skan pada permukaan sel-sel yang

dijangkiti virus rekombinan, pada permukaan protein perlindung virus dan adalah

aktif secara biologikal serta berkebolehan berfungsi. Ciri-ciri fizikal virus yang

berlainan juga telah dikaji dengan bantuan mikroskop electron (EM).

Jangkitan virus rekombinan recHNAF2240 pada fasa log akhir pada m.o.i. 1

merupakan nilai yang paling sesuai untuk penghasilan protein yang optimum.

Penghasilan tindakan keimunan oleh vaksin subunit berdasarkan ujian ill, ELISA

and analisis "Western blot" menunjukkan bahawa temp at vaksin disuntik and

penggunaan dos kedua adalah perlu untuk menghasilkan tindakbalas imun daripada

ayam-ayam yang dikaji. Namun demikian, ujian awal ini memberi maklumat yang

penting tentang persoalan perlindungan ayam daripada jangkitan virus NDV dan

ujian yang lebih menyeluruh adalah digalakan supaya dapat meningkatkan potensi

vaksin rekombinan yang dihasilkan.

XVlll

CHAPTER I

INTRODUCTION

The production of chicken in Malaysia ranks among the highest in the world

in proportion to its population A 1992 survey reported in Asiaweek (20 April, 1994)

on the various poultry producing countries in Asia, put Malaysia in 4th placing after

China, India and Indonesia, the three highest populated countries in Asia

Furthermore, the 1990 livestock statistics prepared by the Department of Veterinary

Services (DVS) showed that the consumption and production of chicken was the

highest among animal products (Figure 1a) The popularity of poultry products lies

mainly on their nutritive values as both poultry meat and eggs production with

regards to energy is simi lar to milk and is low in saturated fats Also, in Malaysia,

the price of chicken among other common food sources appears to be the lowest

(Figure 1b)

The poultry industry in Malaysia was worth 1 5 billion ringgit in 1992 (more

than 55% of the total value of livestocks) and in 1997, it rose to 4 5 bill ion ringgit

With the intensification of the poultry industry, Malaysia is currently self-sufficient

in poultry eggs and meat but at the same time, is exposed to the inevitable increase 10

the prevalence of disease and loses

a)

b)

Figure 1:

"-"-;:;> ... � Q 0 0 "-

T o N l<;OOOO��----� S 100nIOO.U---------�

z o

� MEAT SOURCE

'_LOCAL PRODUCTION I1TOTAL CONSUMPTION I

PRAWNS

BEEF

CHICKEN

0 5 10 15 20 25 30 RINGGIT MALA YSIAIkg

2

Comparison of Production, Consumption and Prices of Local Meat Sources. a) Production and Consumption of Animal Products in 1990 (DVS Livestock Statistics, 1992) and b) Prices of Common Food Source in 1998 (The Star, 1998).

3

The greatest threat in the poultry industry, not only in Malaysia but also in

Asia is Newcastle disease virus (NDV) that is responsible for 3 1 4 outbreak cases

recorded in Malaysia between 1985 to 1987. In 1 976, NDV caused the US a loss of

around 215 mil l ion US dollars wherelse in Malaysia between 1 973 to 1 977, 9% of all

mortality costing the country 3 .8 8 million ringgit annually was due to NDY.

Vaccination appears to be the mam control of poultry viral disease in

Malaysia and other developed as well as developing countries but NDV being

endemic in this region, stil l poses a threat despite routine vaccination programmes

which cost 1 8 to 50 % of the DVS annual budget (1992). It cannot be denied that

current vaccines (live attenuated or kil led) and local feed-delivered vaccines for

vil lage chickens; a common source of NDV epidemics as well as the readily

avai lable poultry health l ike vaccines and production technology from overseas have

contributed towards improving the control of the disease. Nevertheless, these

approaches alone may prove inadequate for Asia's rapid developing poultry industry

to achieve long term stabil ity and self-sustainabil ity.

Therefore, efforts are being made with the aide of the ever advancing

recombinant DNA technology and molecular biology for the development of more

suitable vaccines derived from local NDV strains which would further complement

the needs of the country's poultry industry. The approach would also cater for a

more effective improvement of viral disease control through research by uti l ising

available local resources. The growing interest and appl ication of this technology

has made possible the identification of specific sites on the surface of the virus which

are crucial for inducing protective immune response. Taking advantage of this fact,

4

the production of vaccines from infectious agent which would not replicate as the

pathogenic agent but able to induce immunity without causing any risk of increased

or altered pathogenicity upon exposure to infection may bring about a more effective

vaccIne

In this study, a subunit vaccine was developed through the cloning of the HN

gene from NDV strain AF224 0, isolated from the viral genomic nucleic acid, into a

Baculovirus Expression Vector System (BEVS) The BEVS is capable of expressing

high levels of recombinant viral protein complete with post-translational

modification in insect cells It will also facil itate the study of the HN gene

expression in isolation from the rest of the NDV structural genes Thus the

objectives of this study are -

to isolate the HN gene using peR methods,

2 to clone the HN gene into BEVS,

3 to express the recombinant HN protein in SF9 insect cells, and

4 to determine the functionality of the recombinant protein

CHAPTER II

LITERA TURE REVIEW

Newcastle Disease Virus

Taxonomic Classification

Newcastle disease virus (NDV) is the aetiological agent of the Newcastle

disease, one of the most serious infectious disease of poultry in many parts of the

world including Malaysia The reclassification of virus taxa in 1 993 by the

International Committee in the Taxonomy of Viruses (lCTV) grouped the NDV

species into the Rubulavlrus genus of the Paramyxovmdae family in the order of

Mononegavlrales (Murphy et al., 1 995, Pringle, 1 998) The other genera under the

Paramyxovzrzdae family are the Morblltvlrus and the Paramyxovlrus where NDV

was earlier classified together with mammalian parainfluenza virus type 1 -5 and

mumps virus NDV is the prototype of the genus and is the only member of the

avian paramyxovlrus - 1 (PMV-l) serotype Various other groups which are

serological ly di stinguishable from NDV have been isolated from avian species and

grouped into eight other serotypes called PMV -2 to PMV -9 (Alexander, 1 986)

5

6

History and Distribution of NDV

The first outbreak ofNDV is known to occur in 1926 on the island of Java,

Indonesia called Batavia showing high mortality (Brandly, 1964) At the same

period, Doyle (1927) and Konno et al (1929) (cited in Brandly, 1964) reported a

similar disease in Korea and in NewcastIe-on-Tyne, where the common name of the

disease was coined. Three years later, NDV spread throughout the entire Southeast

Asia (Lancaster, 1966) where it is now endemic (Aini, 1993), Australia (Johnstone,

1931) and India (Brandly, 1964) Within 10 years, ND had been reported in Japan,

Africa and subsequently in various other countries such as the Middle East as well as

in Europe (Lancaster, 1966) Apparently, ND was only first reported in the US in

1944 (Beach, 1944), Canada in 1948 and in South America in the 1950s (cited in

Beard, 1984)

Newcastle Diease Virus and the Malaysia Scenario

In Peninsular Malaysia, the incidence of an ND outbreak was reported at Parit

Buntar, Perak in 1934 causing high mortalities in susceptible chickens with necrotic

lesions, respIratory signs, diarrhoea and eventually death (Whitworth, 1934, cited In

Lim, 1994) Within a few years, it had spread to other states like Selangor, Melaka,

Kedah and Johor (Wallace, 1939, cited in Lim, 1994) In contrast, the virulent ND

is known to be associated with two sources namely wild bird species like psittacine

birds and chickens The highly virulent virus termed as velogenic viscerotropic

Newcastle disease (VVNO) virus is from the earlier source that killed domestic

chickens within 2 to 4 days upon exposure (Lim, 1994) Pearson et al (1975) (cited