UNIVERSITI PUTRA MALAYSIA

INFECTIOUS BURSAL DISEASE VIRUS: ITS GENOMIC PROPERTIES, EVOLUTION, AND INFECTION

TO THE HEAD-ASSOCIATED LYMPHOID TISSUES OF CHICKEN

TAN DO YEW

FPV 2004 13

INFECTIOUS BURSAL DISEASE VIRUS:

ITS GENOMIC PROPERTIES, EVOLUTION, AND INFECTION TO THE HEAD-ASSOCIATED LYMPHOID TISSUES OF CHICKEN

By

TAN DO YEW

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

September 2004

DEDICATION

To my parents, grandma, and siblings.

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

INFECTIOUS BURSAL DISEASE VIRUS: ITS GENOMIC PROPERTIES, EVOLUTION, AND INFECTION

TO THE HEAD-ASSOCIATED LYMPHOID TISSUES OF CHICKEN

By

TAN DO YEW

September 2004

Chairman: Associate Professor Mohd Hair Bejo, Ph.D.

Faculty: Veterinary Medicine

Infectious bursal disease (IBD) is an important immunosuppressive viral disease of

chicken caused by IBD virus (IBDV). There are several strains of IBDV, and

vaccination may not protect the chicken against all strains. Therefore as for control

and prevention effort, it is important to know which strain is present in the field. In

this study, four IBDV isolated from Malaysia had been characterized in detail. The

isolates were B00/73, B00/81, 94230, and 94268. Based on their pathogenicity and

sequence characteristics, these isolates were identified as very virulent strain of IBDV

(vvIBDV). Further analyses of the genetic sequences of 131 IBDV isolates had

provided new insights into the genomic properties of IBDV. These include its bias in

base usage, the avoidance of CpG and TpA dinucleotides, the unique dinucleotide

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pattern of vvIBDV, and its acquisition of VP5 gene using overprinting strategy.

Meanwhile, a better approach in studying the molecular evolution of IBDV was

introduced and termed as “holistic approach of phylogenetic analysis”. The approach

widens the perspective of phylogenetic analysis and reduces error in phylogenetic

inferences. Using this approach, IBDV isolated from Malaysia were shown to share a

common origin with other foreign vvIBDV isolates. In addition, the vvIBDV isolated

from village chicken (94268 isolate) was found to be evolutionary closely related to

the isolates that affected the commercial chickens; indicating infection by vvIBDV is

not limited within the farm boundary. This study also inquired into the cellular

response of head-associated lymphoid tissues (HALT) following intraocular infection

of vvIBDV. The two major lymphoid tissues of HALT are Harderian gland and

conjunctiva-associated lymphoid tissue; in which together, both are indispensable for

the local immunity in the paraocular region. HALT was impaired by vvIBDV

infection at day 3-4 post-inoculation, but swiftly recovered at day 10. The destruction

of HALT, though transient, may interfere with the vaccination programme for other

respiratory diseases and give chance to opportunistic infection to the ocular and

respiratory tract of chicken.

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

VIRUS PENYAKIT BURSA BERJANGKIT: SIFAT-SIFAT GENOMIK, EVOLUSI, DAN JANGKITANNYA

KEPADA TISU LIMFOID BERSEKUTU KEPALA AYAM

Oleh

TAN DO YEW

September 2004

Pengerusi: Profesor Madya Mohd Hair Bejo, Ph.D.

Fakulti: Perubatan Veterinar

Penyakit bursa berjangkit (IBD) ialah sejenis penyakit ayam yang amat penting di

mana ia boleh melumpuhkan sistem imun ayam dan diakibatkan oleh virus IBD

(IBDV). Terdapat beberapa jenis strain IBDV yang berlainan. Vaksinasi terhadap satu

strain tidak semestinya boleh melindungi ayam daripada ancaman strain yang lain.

Oleh yang demikian, adalah mustahak untuk mengetahui strain IBDV yang wujud di

ladang penternakan untuk kawalan dan pencegahan IBD. Dalam kajian ini, empat

IBD yang diasingkan dari Malaysia telah dicirikan secara terperinci. Virus ini ialah

B00/73, B00/81, 94230, dan 94268. Dengan merujukkan kepada keupayaan virus

untuk mengakibatkan penyakit IBD dan jujukan genetiknya, virus ini telah

dikenalpasti sebagai strain amat virulen IBDV (vvIBDV). Penganalisisan selanjutnya

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yang menggunakan 131 jujukan genetik telah memberi gambaran baru terhadap sifat

genomik IBDV. Sifat ini termasuk kecenderungan virus dalam penggunaan nukleotid

tertentu, penghindaran pasangan nukleotid CpG dan TpA, corak unik pasangan

nukleotid vvIBDV, dan pemerolehan gen VP5 dengan menggunakan strategi

pertindihan. Sementara itu, pendekatan kajian evolusi molekul yang lebih baik juga

diperkenalkan dan dinamakan sebagai “Pendekatan keseluruhan dalam analisis

filogenetik”. Pendekatan baru ini dapat meluaskan perspektif analisis filogenetik dan

mengurangkan kesilapan dalam membuat kesimpulan filogenetik. Dengan

menggunakan pendekatan keseluruhan, IBDV yang diasingkan di Malaysia telah

ditunjukkan berasal dari punca yang sama dengan virus IBD negara asing. Di

samping itu, vvIBDV yang diasingkan daripada ayam kampung (virus 94268)

mempunyai hubungan evolusi yang rapat dengan virus yang menjangkit ayam

komersil. Ini menunjukkan jangkitan vvIBDV adalah tidak terbatas kepada ladang

sahaja. Kajian ini juga menyiasat gerak balas tisu limfoid bersekutu di kepala

(HALT) ayam berikutan jangkitan intraokulus oleh vvIBDV. HALT terdiri daripada

dua tisu limfoid yang utama, iaitu kelenjar Harderian dan tisu limfoid bersekutu di

konjunktiva. Kedua-duanya adalah amat diperlukan untuk pelalian setempat di

persekitaran mata. Jangkitan vvIBDV merosakkan HALT pada hari yang ketiga dan

keempat sesudah inokulasi virus. Akan tetapi, HALT pulih dengan cepat pada hari

yang kesepuluh. Pemusnahan HALT, meskipun sementara, mungkin boleh

menjejaskan program vaksinasi terhadap penyakit-penyakit salur pernafasan.

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Tambahan lagi, ini akan memberi kesempatan kepada penyakit-penyakit lain untuk

menjangkit kawasan sekitar okulus dan saluran pernafasan ayam.

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ACKNOWLEDGEMENTS

I would like to thank Associate Professor Dr Mohd Hair Bejo, the chairman of

the supervisory committee for his excellent guidance throughout my PhD programme.

He always willing to take time out of his hectic schedule to discuss the project; and

without fail, gives invaluable comments and suggestions. One of the greatest things a

supervisor could help his or her student is to share the laboratory works. And yes, he

did. He actually teamed up with us (me and other students) during the wearisome

days of samplings for pathogenicity studies. He helped us out in doing post mortem,

taking blood, weighing chickens, evaluating gross lesions…at a glance, one might

think he was one of the students! Indeed, students from other labs envied us for

having such a good supervisor. I would also like to thank him for giving me plenty of

room to probe into other fields, such as bioinformatics and multivariate analysis.

My sincere thanks also go to Professor Aini Ideris and Associate Professor Dr

Rahman Omar, for their constructive suggestions and critics. As the members of the

supervisory committee, at many times, their comments and encouragement had not

only motivated me but also keep things moving in the right track. To Dr Goh Yong

Meng for scrutinizing my first paper before I submitted for peer review; his

intelligence and knowledge truly inspired me. Finally, I would like to thank all my

friends and EM staffs who had helped throughout my PhD programme: Phong,

Chong, Balkis, Nong, Mr Ho, Kong, Khor, Kenny, Karuna, Ali, Wan, Lee, Saiful,

Cheng, Maureen… to name a few. Their friendship is priceless.

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I certify that an Examination Committee met on 13th September 2004 to conduct the final examination of Tan Do Yew on his Doctor of Philosophy thesis entitled “Infectious bursal disease virus: its genomic properties, evolution, and infection to the head-associated lymphoid tissues of chicken” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: ABDUL RANI BAHAMAN, Ph.D. Professor, Faculty of Veterinary Medicine, Universiti Putra Malaysia. (Chairman) FAUZIAH OTHMAN, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) SITI SURI ARSHAD, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) JIMMY KWANG, Ph.D. Professor and Principal Investigator, Temasek Life Science Laboratory, National Universiti of Singapore, (Independent Examiner) ___________________________________

GULAM RUSUL RAHMAT ALI, Ph.D. Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: MOHD HAIR BEJO, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) ABDUL RAHMAN OMAR, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

AINI IDERIS, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) ___________________________________

AINI IDERIS, Ph.D. Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date:

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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.

___________________

TAN DO YEW

Date:

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LIST OF TABLES

Table Page

3.1

Brief histories about the selected four IBDV isolates

3.4

4.1

IBDV isolates used in the sequence analysis 4.14

4.2

Restriction enzymes in relation to the IBDV strains 4.17

4.3

Types of mutation of VP2 gene 4.29

5.1

Base usage percentages and the ranking of serotype 1 IBDV genes (Mean ± SD)

5.12

5.2 Comparisons of hosts and IBDV genomic GC content (Means)

5.18

6.1 Heuristic evaluation of literature on phylogeny study of IBDV

6.4

6.2

IBDV isolates used in the phylogenetic analysis

6.9

6.3 A brief description of PHYLIP modules used in holistic

approach of phylogenetic analysis 6.12

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LIST OF FIGURES

Figure Page

3.1

Gross lesions of the infected specific-pathogen-free chicken embryos

3.10

3.2

Gross lesions of the infected specific-pathogen-free chickens

3.13

3.3

Histological lesions of the infected bursa of Fabricius 3.14

3.4

Electron microscopy of the infected bursal tissue 3.15

3.5

Negative staining and solid phase immune electron microscopy

3.16

4.1

Amplified full-length 1.35 kb IBDV VP2 genes 4.16

4.2

Virtual restriction enzyme analysis of the VP2 gene 4.18

4.3 Sequence identity matrix of VP2 genes of IBDV isolates 4.22

4.4

Number of nucleotide differences in VP2 gene between IBDV isolates

4.23

4.5

Amino acid substitutions of Malaysian IBDV isolates 4.25

4.6

Amino acid substitutions of VP2 gene

4.26

5.1 Cluster analysis of serotype 1 IBDV genes based on nucleotide composition

5.16

5.2

Results from the DF-PCA of IBDV genome 5.25

5.3

The mapping of the hypervariable region of B00/73, B00/81, 94230, and 94268 isolates onto DF-PCA graph (as in Figure 5.2A).

5.30

6.1 Holistic approach of phylogenetic analysis 6.11

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6.2 Phylogenetic analysis of nucleotide sequences of IBDV 6.16

6.3 Phylogenetic analysis of amino acid sequences of IBDV

6.18

7.1

Histology of the Harderian gland

7.12

7.2

Histology of conjunctiva-associated lymphoid tissue

7.13

7.3 Histology of bursa of Fabricius 7.14

7.4

Bursal indices of vvIBDV-infected and non-infected groups

7.17

7.5

Lesion scores of bursa of Fabricius, Harderian gland, and conjunctiva-associated lymphoid tissue

7.18

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LIST OF ABBREVIATIONS

A Adenine AA Amino acid sequences AGID Agar gel immunodiffusion AGPT Agar gel precipitation test atIBDV Attenuated strain of infectious bursal disease virus ayIBDV Atypical strain of infectious bursal disease virus BF Bursa of Fabricius BI Bursal index BP Bootstrap percentage/bootstrap percentages BSA Bovine serum albumin BSNV Blotched snakehead virus C Cytosine CALT Conjunctiva-associated lymphoid tissue CAM Chorioallantoic membrane CAV Chicken anaemia virus cDNA Complementary deoxyribonucleic acid CDS Coding sequence CEF Chicken embryo fibroblast CMI Cell-mediated immunity

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cvIBDV Classical strain of infectious bursal disease virus d Day DAB Diaminobenzidine tetrahydrochloride ddH2O Deionized double-distilled water DEPC Diethyl pyrocarbonate DF-PCA Principal component analysis of dinucleotide frequencies DMSO Dimethylsulphoxide DNA Deoxyribonucleic acid DnI Dinucleotide index dNTP Deoxynucleoside triphosphate dsDNA Double-stranded DNA DTT Dithiothreitol DXV Drosophila X virus ELD50 Fifty percent embryo lethal dose ELISA Enzyme-linked immunosorbent assay G Guanine h Hour HALT Head-associated lymphoid tissues HE Haematoxylin-and-eosin HG Harderian gland HVR Hypervariable region

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IB Infectious bronchitis IBD Infectious bursal disease IBDV Infectious bursal disease virus/Infectious bursal disease viruses IBDV-BDA IBDV-bursal disease antibody IBV Infectious bronchitis virus ICX IBDV immune complex vaccine IPNV Infectious pancreatic necrosis virus IPS Immunoperoxidase staining technique JC69 Jukes and Cantor 1969 model JTT Jones-Taylor-Thornton model K2P Kimura’s two-parameter model LB Luria-Bertani LE Lymphoepithelium ML Maximum likelihood MP Maximum parsimony ND Newcastle disease NDV Newcastle disease virus NT Nucleotide sequences OIE Office international des epizooties ORF Open reading frame PBS Phosphate-buffered saline

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PC Plasma cell/plasma cells PCR Polymerase chain reaction PI Post-inoculation RBC Red blood cells RE Restriction enzyme RNA Ribonucleic acid RT-PCR Reverse transcriptase-polymerase chain reaction SDS Sodium dodecyl sulfate SPF Specific-pathogen-free SPIEM Solid phase immune electron microscopy T Thymine TAE Tris-acetate-EDTA TE Tris-EDTA TNE Tris-natrium-EDTA TEM Transmission electron microscopy UPGMA Unweighted pair-group average vaIBDV Variant strain of infectious bursal disease virus VLP Virus-like particles VN Virus neutralization VNF Virus neutralization factor vvIBDV Very virulent strain of infectious bursal disease virus

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TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xv CHAPTER 1 INTRODUCTION 1.1 2 LITERATURE REVIEW 2.1 2.1 Infectious Bursal Disease (IBD) 2.1 2.1.1 Pathology and Lesions 2.3 2.1.2 Pathogenesis 2.7 2.1.3 Immunosuppression 2.17 2.1.4 Economic Impact 2.20 2.1.5 Epidemiology 2.21 2.2 Infectious Bursal Disease Virus (IBDV) 2.26 2.2.1 Description of the Virus 2.26 2.2.2 Genome and Proteins 2.28 2.2.3 IBDV in Medical Research 2.36

2.3 Diagnosis of IBD 2.37 2.3.1 Clinical and Histopathological Diagnosis 2.37 2.3.2 Serological Diagnosis 2.39 2.3.3 Virological Diagnosis 2.41 2.4 Control and Prevention of IBD 2.44 2.4.1 Exclusion or Eradication 2.44 2.4.2 Genetic Selection for Resistance 2.46 2.4.3 Vaccination 2.46 2.4.4 Anti-viral Drugs 2.58

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3 IN VIVO CHARACTERIZATION OF LOCAL INFECTIOUS BURSAL DISEASE VIRUS ISOLATES 3.1 3.1 Introduction 3.1 3.2 Materials and Methods 3.3

3.2.1 IBDV Isolation 3.3 3.2.2 Experimental Infection in Specific-Pathogen-Free

(SPF) Embryonated Chicken Eggs 3.5 3.2.3 Experimental Infection in SPF chickens 3.6 3.2.4 Histopathology and Electron Microscopy 3.7 3.2.5 Virus Purification and Negative Staining 3.8 3.2.6 Solid Phase Immune Electron Microscopy (SPIEM) 3.8

3.3 Results 3.9 3.3.1 Experimental Infection in SPF Embryonated Chicken Eggs 3.9 3.3.2 Experimental Infection in SPF Chickens 3.11 3.3.3 Gross and Microscopic Lesions 3.11 3.3.4 Electron Microscopy 3.12

3.4 Discussion 3.17 3.5 Conclusion 3.20

4 SEQUENCE ANALYSIS OF VP2 GENE 4.1

4.1 Introduction 4.1 4.2 Materials and Methods 4.4 4.2.1 Sample Preparation for Four IBDV Isolates 4.4

4.2.2 Extraction of Viral RNA 4.4 4.2.3 Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) 4.5 4.2.4 Gel Electrophoresis and Ethidium Bromide Staining 4.7 4.2.5 Purification of RT-PCR Products 4.8 4.2.6 Cloning of the Full-Length VP2 Genes 4.9 4.2.7 Plasmid Extraction and Purification 4.10 4.2.8 Restriction Enzyme Digestion Analysis 4.11 4.2.9 DNA Sequencing 4.12 4.2.10 Sequence Assembly and Analysis using Bioinformatics Software 4.13

4.3 Results and Discussion 4.15 4.3.1 Amplification and Cloning of IBDV VP2 Genes 4.15 4.3.2 Virtual Restriction Enzyme Analysis 4.15 4.3.3 Identification of Amino Acid Markers in VP2 Gene 4.21 4.3.4 Mutations in VP2 Gene 4.21 4.4 Conclusion 4.30

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5 BASE USAGE AND DINUCLEOTIDE FREQUENCY OF IBDV 5.1

5.1 Introduction 5.1 5.2 Materials and Methods 5.6 5.2.1 IBDV Gene Sequences 5.6 5.2.2 Host Coding Sequences and Highly Expressed Genes in the Bursa of Fabricius 5.8 5.2.3 Bioinformatics Software 5.9 5.2.4 Computation of Base Usage and Dinucleotide Frequency 5.9 5.3 Results and Discussion 5.10 5.3.1 Base Usage of Serotype 1 IBDV Genes 5.10 5.3.2 Peculiarity of the Base Usage Pattern in VP5 Gene 5.14

5.3.3 GC Content of IBDV Genome is Similar to the Host 5.17 5.3.4 IBDV Genome is Devoid of CpG and TpA Dinucleotides 5.19 5.3.5 Deficiency of CpG is Correlated with TpG Abundance 5.21 5.3.6 Dinucleotide Patterns are Different Among IBDV strains 5.23 5.3.7 Base Usage and Dinucleotide Frequency of B00/73, B00/81, 94230, and 94268 Isolates 5.29

5.4 Conclusion 5.31 6 MOLECULAR EVOLUTION OF IBDV 6.1

6.1 Introduction 6.1 6.2 Materials and Methods 6.8 6.2.1 IBDV Sequences 6.8 6.2.2 Selection of IBDV Sequences 6.8 6.2.3 Sequence Preparation Prior to Phylogenetic Analysis 6.8 6.2.4 Holistic Approach of Phylogenetic Analysis using PHYLIP 6.10 6.2.5 Editing Phylogenetic Trees 6.12 6.3 Results and Discussion 6.13 6.3.1 Analysis of the Nucleotide Sequences 6.15 6.3.2 Analysis of the Amino Acid Sequences 6.17 6.3.3 IBDV Isolated from Village Chicken (“94268”) was Closely Related with the Isolates that Infected Commercial Chickens 6.20

6.3.4 Holistic versus Conventional Approach of Phylogenetic Analysis 6.21

6.4 Conclusion 6.23

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7 VERY VIRULENT IBDV INFECTION TO THE HEAD-ASSOCIATED LYMPHOID TISSUES OF CHICKEN 7.1 7.1 Introduction 7.1 7.2 Materials and Methods 7.4 7.2.1 Preparation of IBDV Inoculum 7.4 7.2.2 Experimental Infection in HALT of SPF Chickens 7.4

7.2.3 Coating Slides with Adhesive 7.6 7.2.4 Staining and Immunohistochemistry 7.6 7.2.5 Quality Control of Immunohistochemistry 7.7 7.2.6 Lesion Scoring 7.8 7.2.7 Bursal Index 7.8 7.2.8 Software 7.8

7.3 Results 7.9 7.3.1 Clinical Signs, Mortalities, and Gross Lesions 7.9 7.3.2 Histology of Normal Head-Associated Lymphoid Tissues and Bursa of Fabricius 7.9 7.3.3 Infection of vvIBDV to HALT and BF 7.11 7.3.4 The Relationship between Lesions in the BF, HG, and CALT 7.18 7.4 Discussion 7.19 7.4.1 IBDV Infection to the Harderian Gland 7.19 7.4.2 IBDV Infection to the Conjunctiva-Associated Lymphoid Tissue 7.21 7.4.3 IBDV Infection to the Bursa of Fabricius 7.25 7.4.4 The Trio of IBDV Infection: HG, CALT, and BF 7.26 7.4.5 Viral Infection to the HALT – the Implications to Vaccination 7.28 7.5 Conclusion 7.30

8 GENERAL DISCUSSION AND CONCLUSION 8.1

8.1 Status of IBDV in Malaysia 8.1 8.2 Epidemiology of IBD 8.2 8.3 A New Insight into IBDV Genomic Properties 8.2 8.4 A New Insight into the Evolutionary History of IBDV 8.4 8.5 IBDV Infection to the HALT 8.5 8.6 Novel and Improved Techniques 8.6

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REFERENCES/BIBLOGRAPHY R.1 APPENDICES A.1 BIODATA OF THE AUTHOR B.1

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CHAPTER 1

INTRODUCTION

Infectious bursal disease (IBD) is defined, by the Office International des

Epizooties (OIE), as a highly contagious viral disease that destructs the lymphoid

organs of young chickens specifically the bursa of Fabricius, and leads to

immunosuppression and death. IBD topped the list of most serious poultry diseases in

a survey conducted by World Poultry in 1999 (van der Sluis 1999), testifying its

pressing threat to the poultry industry worldwide.

IBD is also known as “Gumboro disease” because it was first reported in

Gumboro, Delaware (USA) by Cosgrove in 1962. Nonetheless, to accurately reflect

the characteristics of the disease, it was later termed “infectious bursal disease” by the

typical lesions found in the infected bursa of Fabricius. The causative agent of IBD is

IBD virus (IBDV), a double stranded RNA (dsRNA) virus in the genus of

Avibirnavirus (Dobos and others 1979). IBDV targets and destructs actively dividing

immature B-lymphocytes (Burkhardt and Muller 1987), in which these cells are

important for the chicken immune system. Therefore, the infected birds are

immunosuppressed because their immune system had been decimated by IBDV.

1.1