OPTIMIZATION OF CELL CULTURE CONDITIONS FOR THE PRODUCTION OF NEWCASTLE DISEASE

VIRUS

BY

MOHD AZMIR BIN ARIFIN

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology

Engineering)

Kulliyah of Engineering International Islamic University

Malaysia

AUGUST 2011 

ii  

ABSTRACT

The present work aims to prepare a model for the production of lentogenic Asplin F strain of Newcastle disease virus (NDV) using cell culture in bioreactor for live vaccine preparation. First NDV was adapted in four different cell lines namely DF-1, CEF, MSB-1 and Vero cells in T-flasks. DF-1 was found to be the most potential for NDV propagation in terms of high NDV production, rapid proliferation rate and several ethical values. Viruses produced in DF-1 cell culture were confirmed as lentogenic NDV strain by molecular diagnosis by using reverse transcriptase polymerase chain reaction (RT-PCR). In a screening experiment, high cell concentration during time of infection was found to be the most significant factor for NDV production. In another screening experiment, DF-1 was cultured in DMEM, DMEM-F12, RPMI 1640 and MEM. Culture of DF-1 cells in DMEM has the highest cell concentration of 1.240 × 106 cells/ml with specific growth rate and doubling time of 0.0180 h-1 and 38.4967 hours respectively. Composition of culture medium was later optimized and it was observed that culture has optimum cell concentration of 1.368 x 106 cells/ml when 2.1 g/L of NaHCO3 and 7.5% serum is included in the culture medium. Experiment was continued with culture of DF-1 cells in spinner vessel. In an optimization of microcarrier concentration study, culture with 3 g/L microcarrier achieved maximum cell concentration of 0.515 × 106 cells/ml compared to culture having 1 g/L, 2 g/L, 4 g/L and 5 g/L microcarriers. Based on this result and previous optimization studies, NDV was propagated in DF-1 cell culture in spinner vessel and the maximum virus titre achieved was 128 HA unit with infectivity titre of 3.08 x 107 TCID50/ml. In a separate experiment using 2-L stirred tank bioreactor, an optimization of process conditions study was conducted and it was analyzed that maximum cell concentration of 1.213 × 106 cells/ml will be obtained when the agitation rate is set at 71 rpm, microcarrier concentration at 2.9 g/L and pO2 pressure at 20%. NDV was also propagated in DF-1 cell culture in stirred tank bioreactor and the maximum virus titre achieved was 4 HA unit with infectivity titre of 1.03 x 103 TCID50/ml. Viruses produced from both types of bioreactors were later concentrated by using the high speed centrifugation method. An optimization study on process conditions of high speed centrifugation was conducted and it was revealed that optimum virus titre of 256 HA unit was achieved when the sample concentration was set at 50%, centrifugation speed was set at 12800 rpm and centrifugation time was set at 8 hours. The ability of cell culture in producing NDV in bioreactors has been shown in this study thus the model can be proposed for ND vaccine production in the future.

iii  

خلاصة البحث

ل ذا العم داد ه ى إع دف إل وذج لإ يه اجنم ن lentogenic F (Asplin (نت لالةم ل س رض نيوآاس روس م في

)NDV .تم تكييف أولاً،) باستخدام تقنية في المفاعل الحيوي لإعداد اللقاح الحي NDV ة سلالة في ة أربع مختلف

ا ن الخلاي ي م ا DF - 1 ، CEF ،MSB - 1 وه ي Vero وخلاي وارير ف دت ل T .DF - 1 ق ر وج ون الأآث تك

يم و السريع الانتشارمعدل عالية ، و NDV من حيث الإنتاج NDV لإآثار المحتملة ة عدة الق وتأآدت .الأخلاقي

ي NDV lentogenicآما سلالة DF - 1الفيروسات التي نتجت من زراعة الخلايا بواسطة التشخيص الجزيئ

ة خلال ).RT - PCRباستخدام تفاعل البلمرة العكسية الناسخ ( ة الخلي في تجربة الفرز ، تبين أن الترآيزات عالي

اج ل لإنت م عام ن أه دوى م رة الع رز آخرى ، زرعت NDVفت ة الف ي تجرب ات DF - 1. ف ي بيئ ، DMEMف

DMEM F12 ،RPMI 1640 وMEM ا ي DF – 1. الخلاي ة ف ن DMEMالمزروع ى م ز أعل ه ترآي لدي

ة و 106 × 1.240خلي دل نم ة / مل مع مع ساعات 38.4967/ ساعة و 0.0180محدد ووقت مضاعفة خلي

ل هي ة الأمث ة البيئ ز خلي وحظ أن ترآي ة المزروعة ول ة البيئ x 1.368على التوالي. بعد ذلك، تم تحسين ترآيب

ة. في مصل الدم٪ 7.5و NaHCO3غرام / لتر من 2.1من خلية / مل عندما يتم تضمين 106 في خلية البيئ

ة أعطي غرام / لتر من 3)، البيئة بترآيز حاملات دقيقة( microcarrierلتحسين ترآيز الدراسة حاملات دقيق

غرام / لتر من 5غرام / لتر و 4غرام / لتر ، 2غرام / لتر ، 1ترآيز أعلى للخلية عند مقارنة مع البيئة بترآيز

ذه النتيجة ودراسات خ 106 × 0.515بترآيز خلية حققت الحد الأقصى حاملات دقيقة ى ه تنادا إل لية / مل. واس

روس DF - 1تكاثرت في الخلية الزارعة NDVالتحسين السابقة، ار الفي دوار و الحد الأقصى لعي في وعاء ال

ة منفصلة باستخدام .x 107 TCID50/ml 3.08مع عيار العدوى من HAوحدة 128الذي تحقق هي في تجرب

ا بحصول الحد الأقصى متحركلتر خزان مفاعل حيوي 2 م تحليله ، وأجريت دراسة لتحسين ظروف العملية وت

ة 106 × 1.213للترآيز من خلايا ز دوره. 71خلية / مل عند تعيين معدل الهياج في الدقيق ة ترآي حاملات دقيق

اثرت ٪ 20بنسبة الضغط PO2و / لتر غرام 2.9 ة في NDV. تك ة بيئ وي في خزان DF - 1 خلي مفاعل حي

روس عيار أقصى خزان ومتحرك ذي تحقق الفي ة مع HA وحدة 4 أقصى ال ار من العدوى مقارن x 1.03 عي

103. TCID50/ml . ة ترآزت في وقت لاحق الفيروسات التي الناتجة من آلا النوعين من المفاعلات البيولوجي

ين ظروف العم ة لتحس ت دراس د أجري ائق. وق ذ ف لوب تنبي تخدام الأس ل باس ار الأمث ين أن عي ائق وتب ذ ف ة لتنبي لي

ة بنسبة HA 256الفيروس تحقق وحدة ز العين ين ترآي م تعي ذ في 50عندما ت دورة في 12800٪ ، سرعة تنبي

اج 8الدقيقة، وآان الوقت المحدد الطرد في ا في إنت ة زراعة الخلاي درة تقني ساعات. وقد أظهرت هذه الدراسة ق

NDV في المفاعلات الحيوية في وبالتالي يمكن اقتراح نموذج لإنتاج لقاحND .في المستقبل

iv  

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology Engineering).

_______________________ Maizirwan Mel Supervisor

_______________________ Mohamed Ismail Abd. Karim Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology Engineering).               ___________________________ 

Examiner (Internal) Raha Ahmad Raus

________________________ Examiner (External) Fadzilah Adibah Abdul Majid

This thesis was submitted to the Department of Biotechnology Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering).

________________________ Md. Zahangir Alam Head, Department of Biotechnology Engineering

This thesis was submitted to the Kulliyyah of Engineering and is accepted as a fulfillment of the requirement for the degree of Master of Science (Biotechnology Engineering).

________________________ Amir Akramin Shafie Dean, Kulliyyah of Engineering

v  

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where

otherwise stated. I also declare that it has not been previously or concurrently

submitted as a whole for any other degrees at IIUM or other institutions.

Mohd Azmir Bin Arifin

Signature ……………………………………. Date …………………………….

vi  

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright © 2011 by Mohd Azmir Bin Arifin. All rights reserved.

OPTMIZATION OF CELL CULTURE CONDITIONS FOR THE PRODUCTION OF NEWCASTLE DISEASE VIRUS

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research

may only be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies

(print or electronic) for institutional and academic purposes. 3. The IIUM library will have the right to make, store in a retrieval

system and supply copies of this unpublished research if requested by other universities and research libraries.

Affirmed by: Mohd Azmir Bin Arifin

………………………….. …………………… Signature Date

 

vii  

ACKNOWLEDGEMENTS

Praises to Allah S.W.T., The Almighty God for His blessings has enabled me to accomplish the study. I would like to express the deepest appreciation to my supervisor, Assoc. Prof. Dr. Maizirwan Mel for his guidance, advises, encouragement, patience and valuable assistance that enable me to accomplish MSc. program smoothly and efficiently. I also would like to thank my co-supervisors, Prof. Mohd Ismail Abd. Karim and Assoc. Prof. Dr. Sharifah Syed Hassan for their invaluable input, time and support during my study.

I sincerely appreciate the friendship and assistance from all lab mates and lab technicians, Br. Jaafar, Sr. Siti Hajar, Sr. Yusilawati, Sr. Najiah, Sr. Alawiyah, Br. Tamrin, Br. Zulkarnain, Br. Wan Fazli, Br. Saiful, Br. Hafizul, Br. Aslan and Br. Sukiman. My special thanks go to En. Kamaruddin who had shared my burden and worked restlessly in adapting Newcastle disease virus in various cell lines. I am deeply grateful to his commitment and assistance. I wish to extend my appreciation to everyone, although not individually named here, who had contributed directly or indirectly to my project and thesis.

Last but not least, I want to take the opportunity to thank my parents, brothers

and sisters for their endless love, care and encouragement. Without all of you, it would not be possible for me to complete my project and thesis. May Allah bless you all for your kindness.

viii  

TABLE OF CONTENTS

Abstract ii Abstract in Arabic iii Approval Page iv Declaration Page v Copyright Page vi Acknowledgements vii Table of Contents viii List of Tables xii List of Figures xiv List of Abbreviations xvi

CHAPTER1: INTRODUCTION 1 1.1 Background 1 1.2 Problem Statement and its Significance 3 1.3 Research Hypothesis 4 1.4 Research Objectives 5 1.5 Research Methodology 5 1.6 Scope of Study 7 1.7 Thesis Organization 7

CHAPTER 2: LITERATURE REVIEW 8 2.1 Introduction 8

2.1.1 Host Range 9 2.1.2 Pathotypes 9 2.1.3 Transmission 11

2.2 Newcastle Disease Virus 11 2.2.1 Classification 11 2.2.2 Virion Composition 12 2.2.3 Morphology 13 2.2.4 Genome Organization 15 2.2.5 Nucleocapsid and Its Associated Protein 16

2.2.5.1 Nucleocapsid Protein (NP) 16 2.2.5.2 Phosphoprotein (P) 17 2.2.5.3 Large Protein (L) 17

2.2.6 Matrix Protein (M) 18 2.2.7 Envelope Glycoproteins 18

2.2.7.1 Haemagglutinin-neuraminidase Protein (HN) 19 2.2.7.2 Fusion Protein (F) 19

2.2.8 Stages of Replication of NDV 20 2.2.8.1 Virus Attachment, Fusion and Entry 21 2.2.8.2 Transcription 21 2.2.8.3 Replication 22 2.2.8.4 Virus Assembly and Release 23

2.3 Newcastle Disease Vaccines 25

ix  

2.3.1 Live Lentogenic Vaccines 26 2.3.2 Live Mesogenic Vaccines 26 2.3.3 Inactivated Vaccines 27

2.4 Animal Cell Culture 27 2.4.1 Type of Cell Cultures 29

2.4.1.1 Primary Cell Culture 29 2.4.1.2 Finite Cell Lines 31 2.2.1.3 Continuous Cell Lines 32

2.4.1.3.1 DF-1 Cell Line 33 2.4.1.3.2 Vero Cell Line 34 2.4.1.3.3 MSB-1 Cell Line 35

2.4.2 Mode of Culture 35 2.4.2.1 Anchorage Dependent Cell Culture 35 2.4.2.2 Suspension Cell Culture 36

2.4.3 The Phases of Culture 37 2.4.3.1 Lag Phase 38 2.4.3.2 Exponential Phase 39 2.4.3.3 Stationary Phase 40 2.4.3.4 Decline Phase 40

2.4.4 Culture Medium 41 2.4.4.1 Water 42 2.4.4.2 Glucose 42 2.4.4.3 Amino Acids 43 2.4.4.4 Vitamins 43 2.4.4.5 Salts 44 2.4.4.6 Antibiotics 44 2.4.4.7 Buffering System 45 2.4.4.8 Serum 46

2.4.5 Bioreactor for Animal Cells 47 2.4.5.1 Homogeneous Bioreactors 47

2.4.5.1.1 Spinner Vessel 48 2.4.5.1.2 Stirred Tank Bioreactor 49

2.4.5.2 Heterogeneous Bioreactors 49 2.4.5.3 Factors Affecting Cell Culture in Bioreactor 50

2.4.5.3.1 Aeration 50 2.4.5.3.2 Agitation 51

2.4.5.4 Microcarrier Cell Culture Technology 52 2.5 Virus Culture 53

2.5.1 Virus Cultivation in Embryonated Chicken Eggs 54 2.5.2 Virus Cultivation in Cell Culture 55 2.5.3 Detection of Viruses and Virus Components 57

2.5.3.1 Detection of Virions 57 2.5.3.2 Detection of Infectivity Using Cell Cultures 58 2.5.3.3 Detection of Virus Nucleic Acids 59

2.5.4 Infectivity Assay 59 2.5.4.1 Quantitative Assay 59 2.5.4.2 Quantal Assay 60

2.5.5 Physical Assay 61 2.5.6 Virus Concentration 62

x  

2.5.6.1 Sonication 63 2.5.6.2 Ultracentrifugation 64

2.6 Summary 64 CHAPTER 3: MATERIALS AND METHODS 66

3.1 Introduction 66 3.2 Materials 66

3.2.1 Virus Strains 68 3.2.2 Cell Lines 68 3.2.3 Culture Medium 68 3.2.4 Microcarriers 68 3.2.5 Bioreactor Systems 69 3.2.6 Chemicals and Reagents 69 3.2.7 Equipments 69 3.2.8 Consumable Items 69

3.3 Methods 70 3.3.1 Medium Preparation 70 3.3.2 Thawing of Cryopreserved Cells 70 3.3.3 Subculturing 71 3.3.4 Microcarrier Preparation 71 3.3.5 Cell Growth in Bioreactor Systems 72

3.3.5.1 T-flask Culture 72 3.3.5.2 Spinner Vessel 72 3.3.5.3 Stirred Tank Bioreactor 73

3.3.6 Cell Counting in Different Bioreactors 73 3.3.6.1 T flask Culture 74 3.3.6.2 Spinner Vessel and Stirred Tank Bioreactor 75

3.3.7 Specific Growth Rate and Doubling Time 76 3.3.8 Cell Infection with NDV in Different Bioreactors 77

3.3.8.1 T-flask 77 3.3.8.1.1 Harvesting of Virus 77 3.3.8.1.2 Adaptation of Virus 77

3.3.8.2 Spinner Vessel 78 3.3.8.3 Stirred Tank Bioreactor 78

3.3.9 Identification and Confirmation of Virus 79 3.3.10 High Speed Centrifugation 79 3.3.11 Experimental Design 80

3.3.11.1 Screening Important Parameters of NDV Culture in Spinner Vessel 80

3.3.11.2 Optimization of Medium Composition in T-flask 81 3.3.11.3 Optimization of Process Conditions in Stirred Tank Bioreactor 82 3.3.11.4 Optimization of Process Conditions in

High Speed Centrifugation 83 3.3.12 Virus Analysis 84

3.3.12.1 Haemagglutination Assay 84 3.3.12.2 TCID50 Assay 85 3.3.12.3 MOI Determination 85

3.4 Summary 85

xi  

CHAPTER 4: RESULTS AND DISCUSSION 87 4.1 Introduction 87 4.2 Screening and Identifying Potential Host Cell Line for Newcastle Disease Virus (Asplin F Strain) Adaptation 87 4.3 Confirmation of Virus Sample by Using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 92 4.4 Comparative Study: Culturing Other Strains of Newcastle Disease Virus in DF-1 Cells 93 4.5 Screening of Significant Factors of Newcastle Disease Virus Production in Cell Culture 96 4.6 Screening for Best Culture Medium for Growth of DF-1 Cells 100 4.7 Optimization of Medium Composition in T-flask 102 4.8 Virus Growth in Different Bioreactors 108

4.8.1 Optimization of Microcarrier Concentrations in Spinner Vessel 108 4.8.2 Production of Newcastle Disease Virus in Spinner Vessel 111 4.8.3 Optimization of Process Conditions in 2-L Stirred Tank Bioreactor 112 4.8.4 Production of Newcastle Disease Virus in 2-L Stirred Tank Bioreactor 120

4.9 Optimization of Process Conditions of Virus Concentration Using High Speed Centrifugation 123 4.10 Summary 128

CHAPTER 5: CONCLUSIONS AND RECOMMENDATION 129 5.1 Conclusion 129 5.2 Recommendation 131

BIBLIOGRAPHY 133

PUBLICATIONS 151

AWARDS 154

PATENT 156

APPENDIX A DIFFERENT BIOREACTORS 157 APPENDIX B DF-1 CELLS’ MORPHOLOGY ON MICROCARRIERS 159 APPENDIX C DIFFERENT MEDIA COMPOSITION 162 APPENDIX D CELL LINE DATA SHEET 170 APPENDIX E PRIMER SEQUENCE 171

xii

LIST OF TABLES

Table No. Page No.

3.1 2**(3-0) Design for NDV Culture in Spinner Vessel Experiment. 81

3.2

3.3

3**(2-0) Full Factorial Design for T-flask Experiment. 3**(3-1) Fractional Factorial Design for Bioreactor Experiment.

82

83

84

3.4 3**(3-1) Fractional Factorial Design for High Speed Centrifugation.

4.1 Haemagglutination (HA) titre of NDV (Asplin F strain) adaptation

in different cell lines.90

94

96

100

104

105

109

112

113

114

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

4.10

Haemagglutination (HA) titre of different strains of NDV passaged in DF-1 cell culture.  Haemagglutination (HA) titre of NDV (Asplin F strain) produced using different culture conditions in spinner vessel.  Comparison of maximum cell concentration, specific growth rate and doubling time of DF-1 cell in different culture medium.  Experimental design using 3**(2-0) full factorial design (FD) with experimental and predicted (using model equation) values of maximum cell concentration for medium optimization. Analysis of Variance (ANOVA) for Response Surface Quadratic Model for medium optimization. Maximum cell concentration achieved in the culture of DF-1 cell in spinner vessel at different concentrations of microcarriers.  Cell concentration and virus titre produced by DF-1 cell culture in spinner vessel.  Experimental design using 3**(3-0) fractional factorial design (FFD) with experimental and predicted (using model equation) values of maximum cell concentration for process optimization. Analysis of Variance (ANOVA) for Response Surface Quadratic Model for process optimization.

xiii

4.11

4.12

4.13

Cell concentration and virus titre produced by DF-1 cell culture in stirred tank bioreactor.  Experimental design using 3**(3-0) fractional factorial design (FD) with experimental and predicted (using model equation) values of virus titre for process optimization in ultracentrifugation. Analysis of Variance (ANOVA) for Response Surface Quadratic Model for process optimization in ultracentrifugation.

121

124

125

xiv

LIST OF FIGURES

Figure No. Page No.

1.1

2.1

2.2

2.3

2.4

2.5

2.6

3.1

3.2

4.1

4.2

4.3

4.4

4.5

4.6

4.7

Production of Newcastle disease virus in cell culture system Schematic diagram of Newcastle disease virus particle. Electron micrograph of negatively stained pleomorphic Newcastle disease virus (strain Beaudette C) particles obtained from supernatant of infected chicken embryo fibroblast cells. Genetic map of genomic RNA of NDV. Schematic diagram of Paramyxovirus transcription and replication. The life cycle of NDV. Phases of growth in cell culture. An overview of experimental studies. Counting grid of haemacytometer. Growth profile of DF-1 cell in different culture medium. Different cell lines used for NDV (Asplin F Strain) adaptation. (A) DF-1; (B) CEF; (C) Vero; (D) MSB-1. Cytopathic effects shown in different cell lines. (A) DF-1; (B) CEF; (C) Vero. Gel analysis of the 231 bp PCR products were resolved on 1.5% (w/v) agarose gel and stained with ethidium bromide. Lane 1, 100 bp step ladder; lane 2, positive control (UPM); lane 3, positive control (MVP); lane 6, 8th passage of NDV in DF-1; lane 7, 5th passage of NDV in DF-1. DF-1 cell cultures showing CPE after infection by different strains of NDV. (A) La Sota; (B) Mukteswar S; (C) AF 2240. Growth profile of DF-1 cell culture in spinner vessel and production of NDV. Pareto’s chart showing the effects of size of cell inoculums, serum concentration during virus replication phase and MOI on NDV titre produced in spinner vessel.

6

13

14

16

23

25

41

67

75

88

90

91

93

94

97

98

xv

4.8

4.9

4.10

4.11

4.12

4.13

4.14

4.15

4.16

4.17

4.18

4.19

4.20

4.21

Growth profile of DF-1 cell in different culture medium. 3D response surface and 2D contour plots shows the effect of NaHCO3 concentration (g/L) and serum concentration (%) on maximum cell concentration.  Growth profile of DF-1 cell in different compositions of culture medium.  Growth profile of DF-1 cell in spinner vessel at different microcarrier concentrations.  Growth profile of DF-1 cell culture in spinner vessel and production of NDV. Plot of observed versus predicted values for maximum cell concentration. 3D response surface and 2D contour plots shows the effect of agitation rate (rpm) and microcarrier concentration (g/L) on maximum cell concentration.  3D response surface and 2D contour plots shows the effect of agitation rate (rpm) and partial O2 pressure (%) on maximum cell concentration. 3D response surface and 2D contour plots shows the effect of microcarrier concentration (g/L) and partial O2 pressure (%) on maximum cell concentration. Growth profile of DF-1 cell in different process conditions. Growth profile of DF-1 cell culture in stirred tank bioreactor and production of NDV. 3D response surface and 2D contour plots shows the effect of sample concentration (%) and centrifugation speed (rpm) on virus titre. 3D response surface and 2D contour plots shows the effect of sample concentration (%) and centrifugation time (hour) on virus titre. 3D response surface and 2D contour plots shows the effect of centrifugation speed (rpm) and centrifugation time (hour) on virus titre.

101

107

107

109

111

115

117

117

118

119

121

126

126

127

xvi

LIST OF ABBREVIATIONS

ANOVA APMV-1 ATCC ATP BCEFi BHK CAV CEF CPE CS DCU DEAE DF-1 DMEM DMEM/F12 DNA DOE ECACC EDTA EID50 ELISA ELL F FBS FD FLFAM FFD G GE HA HCEFi HeLa HEPES HIV HN HS IFN L LD50 M MDBK MDCK MDV MEM

Analysis of Variance Avian Paramyxovirus Serotype 1 American Type Culture Collection Adenosine Triphosphate Breast-derived CEF Immortal Baby Hamster Kidney Chicken Anemia Virus Chicken Embryo Fibroblast Cytopathic Effect Calf Serum Digital Control Unit Diethylaminoethyl Douglas Foster Dulbecco’s Modification of Eagle’s Medium Dulbecco’s Modification of Eagle’s Medium Formulation-12 Deoxyribonucleic Acid Design of Experiment The European Collection of Cell Cultures Ethylenediaminetetraacetic Acid 50% Egg Infective Dose Enzyme-linked Immunosorbent Assay East Lansing Line Fusion Protein Fetal Bovine Serum Full Factorial Design Federation of Livestock Farmer's Associations of Malaysia Fractional Factorial Design Guanine Gene End Haemagglutination Assay Heart-derived CEF Immortal Human Cervical Carcinoma N-2-Hydroxyethylpiperazine-N’-2-ethane Sulfonic Acid Human Immunodeficiency Virus Haemagglutinin/Neuraminidase Protein Horse Serum Interferon Large Protein 50% Lethal Dose Matrix Protein Madin-Darby Bovine Kidney Madin-Darby Canine Kidney Marek Disease Virus Minimum Essential Medium

xvii

MMR MOI MVP ND NDV NIH NVND OIE ORF P PBS PCR PEG PFU PTFE RBC RDRP RNA RPMI RT-PCR SPF TBE TCID50 UPM UV VP-SFM VRI

Measles, Mumps, and Rubella Multiplicity of Infection Malaysia Vaccine Pharmaceuticals Sdn. Bhd. Newcastle Disease Newcastle Disease Virus National Institute of Health Neurotropic Velogenic Newcastle Disease Office de Internationale Epizooties Open Reading Frame Phosphoprotein Phosphate Buffered Saline Polymerase Chain Reaction Polyethylene Glycol Plaque Forming Unit Polytetrafluoroethylene Red Blood Cells RNA-dependent RNA Polymerase Ribonucleic Acid Rosewell Park Memorial Institute Reverse Transcriptase Polymerase Chain Reaction Specific Pathogen Free Tris/Borate/EDTA 50% Tissue Culture Infective Dose Universiti Putra Malaysia Ultraviolet Virus Production Serum Free Medium Veterinary Research Institute

1  

CHAPTER ONE

INTRODUCTION 1.1 BACKGROUND

Newcastle disease (ND) caused by Newcastle disease virus (NDV) is regarded as one

of the most important viral diseases of poultry in the world (Spradbrow, 1987; Adene,

1990). The disease has a worldwide distribution, and is a major threat to the poultry

industries due to the huge economic loss associated with it. ND which had its first

outbreak in 1926, in Java, Indonesia (Kraneveld, 1926), was named after a place in

England where it was rediscovered a year later (Doyle, 1927). About 27 of the 50

orders of birds have been reported to be susceptible to natural or experimental

infections of NDV (Yusoff and Tan, 2001). Chickens are the most susceptible host, in

which the severity of the disease may vary from mild infection with no apparent

clinical signs to a severe form causing 100% mortality (Huang, et al., 2003).

NDV which is also designated as avian paramyxovirus 1 (APMV-1) is a

member of the genus Avulavirus of the family Paramyxoviridae, in the order

Mononegavirales (Mayo, 2002). Other important pathogens included in this family are

the mumps virus, human parainfluenza virus, sendai virus, simian virus 5 and recently

emerging nipah and hendra viruses. Electron microscopic examinations reveal that

virus particles of members of this group have pleomorphic structure (Yusoff and Tan,

2001). They generally appear as rounded particles with diameters around 100-500 nm

but often filamentous forms of about 100 nm across and variable length are seen

(Alexander, 1988b).

2  

The virus is enveloped and contains a single linear strand, non-segmented

negative sense RNA molecule of 15,186 nucleotides (nt) as its genome

(Krishnamurthy and Samal, 1998; de Leeuw and Peeters, 1999). The genomic RNA of

the virus is consists of six genes, which encode for at least eight proteins (Peeples,

1988). The genes are arranged in tandem in the order of 3’ -NP-P-M-F-HN-L- 5’

which encode for nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M),

fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large polymerase

protein (L), respectively (de Leeuw and Peeters, 1999).

Based on the clinical signs and severity of disease produced in chickens,

strains of NDV are classified into three major pathotypes; lentogenic, mesogenic, and

velogenic. Lentogenic strains cause mild or avirulent infections that are largely limited

to the respiratory. Strains of intermediate virulence that cause respiratory disease with

moderate mortality are termed mesogenic while strains that are highly virulent causing

100% mortality in chickens are termed as velogenic. Velogenic strains can be further

categorized into two types: viscerotropic and neurotropic. Viscerotropic velogenic

strains produce lethal hemorrhagic lesions in the digestive tract whereas neurotropic

velogenic strains produce neurological and respiratory disorders (Spradbrow, 1987;

Alexander, 1997).

NDV is usually transmitted through ‘traditional routes’ such as direct animal

contact, contaminated feed, water, implements, transport contact or transmission by

people (Alexander, 1988a). Airborne transmission is also considered as one of the

important routes for spreading of ND. ND outbreaks in England in 1970–1971 (Hugh-

Jones et al., 1973) and epidemic in Northern Ireland in 1973 (McFerran, 1989) was

believed to be caused by airborne NDV. The success of NDV transmission through

this route depends on many environmental factors, such as temperature, humidity, and

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stocking density. Delay et al. (1948) and Hietala et al. (2005) have demonstrated that

NDV can be recovered from poultry environment air samples. Although airborne

NDV is a major threat to poultry farms, they are usually heat labile and readily

destroyed by exposure to high temperature (>50°C) and also by ultraviolet rays in

sunlight (Lomniczi, 1975).

1.2 PROBLEM STATEMENT AND ITS SIGNIFICANCE

ND has produced huge economic losses to many economies worldwide. Before the

emergence of the highly pathogenic Asian H5N1 influenza virus, the economic impact

of ND has remained unsurpassed by other poultry viruses. In developed countries

where the poultry industries are well established, not only outbreaks of ND are

extremely costly but preventive measures taken to avoid the disease also represent

continuing losses to the industry (Alexander and Senne, 2008). In 2002, an outbreak

caused by a virulent strain of NDV in California, USA has caused $200 million worth

of losses from the depopulation of birds (Kapczynski and King, 2005). Furthermore

countries with ND free status also have to bear huge costs from repeated testing to

maintain the status and for purposes of trade. While in many developing countries,

recurring outbreaks has made ND a major limiting factor to the growth of commercial

poultry industries and establishment of trade links (Alexander and Senne, 2008).

Yusoff (2008) has reported that in Malaysia alone, the losses caused by ND amount to

over RM 100 million in costs annually.

Until now, there is no treatment or method to eradicate ND. Prevention is by

importing birds from disease free flocks or through vaccination that must continue

throughout the life of the bird (Alexander, 1992; OIE, 2002). Traditionally, ND

vaccines have been produced by growing vaccine virus strains in embryonated

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chicken eggs. NDV is harvested from the allantoic fluid and processed to create a

vaccine (Gallili and Nathan, 1998). This traditional method however poses some

drawbacks, such as poor quality control, high labor-intensity, time consuming, needs

high amount of specific pathogen-free eggs, and requires big area for the incubation of

eggs. Besides the process is slow and difficult to scale-up, so large strategic stocks

must be kept to respond in cases of epidemics (Souza, et al., 2009).

In Malaysia there are only two agencies that produce vaccines for ND; the

Veterinary Research Institute (VRI) and also the Malaysia Vaccine Pharmaceuticals

(MVP) Sdn. Bhd. Both agencies use the cumbersome embryonated chicken eggs

method which has resulted to insufficient amount and types of vaccines required to

provide immunities to almost 516 million birds (broilers) produced in Malaysia in

2009 (FLFAM, 2010). To fulfill high local demand of ND vaccines, Malaysia has to

import vaccines from overseas and this step poses a great risk because newer strains

may be introduced to the local poultry industries.

1.3 RESEARCH HYPOTHESES

One method that has high possibility to overcome all the problems mentioned is by

producing ND vaccines by using animal cell culture. Animal cell culture offers many

advantages over the traditional chicken eggs method. The method is rapid, convenient,

less expensive than eggs, supports easy scale up and also it allows evidence of viral

proliferation to be examined microscopically. Many cell substrate systems have been

reported to be able to support the growth of NDV (de Leeuw and Peeters, 1999;

DiNapoli, et al., 2007; Ravindraa, et al., 2008). In addition, more mechanized cell

culture methodology allows virus particles to be produced with the highest control and

quality. There are also numerous types of culture medium and bioreactor systems that

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are easily modified to mimic the environment of virus replication in eggs. And finally

with the advent of microcarrier cell culture technology, high density cell culture is

achievable and virus yield produced from the cell culture is expected to be more than

the embryonated chicken eggs method or at least on par.

1.4 RESEARCH OBJECTIVES

The general objective of this study is to develop a process for producing Newcastle

disease virus (Asplin F strain) for preparation of live vaccine by using cell culture as

an alternative to the traditional embryonated chicken eggs method. The specific

objectives based on the reasons discussed are as follows:

i. To determine the most suitable host cell line for Newcastle disease virus

adaptation.

ii. To optimize Newcastle disease virus production using cell culture in

bioreactor.

iii. To optimize the process conditions for Newcastle disease virus

concentration using the high speed centrifugation method.

1.5 RESEARCH METHODOLOGY

Major steps involved in this study are presented in Figure 1.1. A more detailed

methodology is described in chapter three of this thesis.

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Figure 1.1: Production of Newcastle disease virus in cell culture system

Screening and identifying potential host cell line for Newcastle disease virus (Asplin F strain) adaptation 

Comparison of propagation ability of other strains of Newcastle disease

virus in DF-1 cell culture 

Screening for critical process components of Newcastle disease

virus production in cell culture 

Optimization of medium composition in T-flask for maximum cell yield 

Screening for best culture medium for growth of DF-1 cells 

Optimization of process conditions in spinner vessel and 2-L stirred tank bioreactor for maximum cell yield 

Production of Newcastle disease virus in spinner vessel and 2-L stirred

tank bioreactor 

Optimization of process conditions for virus concentration using the high

speed centrifugation method 

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1.6 SCOPE OF STUDY

The study was confined within the scope of works starting from the adaptation of

Newcastle disease virus in cell culture using T-flasks to concentration of virus

produced from cell culture in spinner vessel and 2-L stirred tank bioreactor by using

the high speed centrifugation method. The outline of the scope is sequentially

arranged as follows:

i. Adaptation of Newcastle disease virus (Asplin F strain) in several cell

lines in T-flasks 

ii. Medium and process optimization for maximum virus production 

iii. Production of Newcastle disease virus in spinner vessel and 2-L stirred

tank bioreactor 

iv. Concentration of virus by using high speed centrifugation 

1.7 THESIS ORGANIZATION

Chapter 1 presents the background, hypotheses, scope, and objectives of the research

work. Chapter 2 reviews the available literature in the field of Newcastle disease virus

production techniques. A detailed review on Newcastle disease and Newcastle disease

virus has been discussed. This chapter also discusses different cell lines used in

Newcastle disease virus propagation as well as the materials and equipments used in

the current study. Chapter three discusses the detailed methodology of the study

including materials and experimental and analytical procedures. Chapter four presents

the experimental results and extensive discussion is given on the basis of results.

Chapter five provides general conclusions of the research work coupled with some

recommendations on how to improve the research.