universiti putra malaysia transcriptome and …psasir.upm.edu.my/43017/1/ib 2012 17r.pdf ·...

UNIVERSITI PUTRA MALAYSIA

MOHAMMAD SYAMSUL REZA BIN HARUN

IB 2012 17

TRANSCRIPTOME AND APOPTOSIS ANALYSIS OF FELINE INFECTIOUS PERITONITIS VIRUS-INFECTED CRANDELL

REES FELINE KIDNEY CELLS

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TRANSCRIPTOME AND APOPTOSIS

ANALYSIS OF FELINE INFECTIOUS

PERITONITIS VIRUS-INFECTED CRANDELL

REES FELINE KIDNEY CELLS


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2012

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

TRANSCRIPTOME AND APOPTOSIS ANALYSIS OF FELINE INFECTIOUS PERITONITIS VIRUS INFECTED CRANDELL REES

FELINE KIDNEY CELLS

By


August 2012

Chair: Professor Dr. Abdul Rahman bin Omar, PhD

Faculty: Institute of Bioscience

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP

virus (FIPV), a virulent mutant of apathogenic feline enteric coronavirus

(FECV). There are no effective diagnostic, vaccine and treatment available

because the virus virulence determinants and pathogenesis are not fully

understood. This study aims to elucidate the early host genes and the

phenomenon of apoptosis associated with in vitro infection of FIPV in cell

culture. RNA samples from FIPV serotype II strain 79-1146 infected Crandell

Rees Feline Kidney (CRFK) cells at 3 hours post infection were sequenced

using Illumina™ next generation sequencer platform then subsequently

analysed with CLC bio Genome Workbench software. Sequencing reads

were mapped to Felis catus 2X annotated shotgun reference genome and

control versus infected cell reads expression analysis was conducted. Kal’s Z-

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test on expression proportions was used to determine significantly expressed

genes. Genes expressed with false discovery rate (FDR) less than 0.05 and

more than 1.99 fold change were considered for further analysis. RNA-seq

analysis mapped both control and infected cell reads to 18899 genes out of

19046 annotated, while expression analysis revealed 61 genes were

differentially expressed by both samples with 44 genes were up regulated

while the rest were down-regulated. Among the genes is a chemokine for

attracting monocytes, CCL8 that was expressed only in infected sample

suggesting that early response against FIPV involves cell mediated immunity

(CMI). In addition, 4 genes (CXCL10, PHF11, ATF3, IRF1) that associated

with Th1 cytokines secretion were also up regulated in this study.

Meanwhile, anti-apoptotic gene RNF7 and ribosomal gene RPL39 were

expressed only in control sample indicating that FIPV initiate apoptosis and

disturb host cell protein translation as early as 3 hours after infection. Besides

that, 9 pro-apoptotic genes (CXCL10, MX1, RSAD2, UBA7, RNF19B, ESE1,

BAK1, CASP7, PD-L1) were up regulated while another 3 anti-apoptotic

genes (c-Kit, CKS2, ID-1) were down regulated. The detail role of those genes

and other differentially expressed genes are discussed. The ability of the

virus to induce apoptosis in CRFK cells was also analysed within 48 hours at

12 different time frames which are 3, 9, 12, 15, 18, 21, 24, 27, 30, 36, 42 and 48

hours by flow cytometry and annexin-V FITC staining. Apoptosis analysis

confirmed that a significant number of cells undergo early apoptosis 18

hours post-infection and late-apoptosis 30 hours post-infection. This study

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has succesfully identified several candidate genes that may play important

role in FIPV pathogenesis and has characterized important events in cell

death following FIPV infection in CRFK cells.

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

ANALISIS TRANSKRIPTOM DAN APOPTOSIS KE ATAS SEL GINJAL KUCING CRANDELL REES YANG DIJANGKITI VIRUS PERITONITIS

BERJANGKIT FELIN

Oleh


Ogos 2012

Pengerusi: Profesor Dr. Abdul Rahman bin Omar, PhD

Fakulti: Institut Biosains

Peritonitis berjangkit felin (FIP) adalah penyakit sistemik yang mematikan

disebabkan oleh virus FIP (FIPV), mutan virulen daripada virus korona

kucing enterik (FECV) yang tidak berbahaya. Tiada terdapat diagnostik,

vaksin dan rawatan yang berkesan kerana faktor penentu virulensi virus dan

patogenesis tidak difahami sepenuhnya. Penyelidikan ini bertujuan untuk

mengkaji gen awal sel CRFK yang diaktifkan semasa jangkitan untuk

menjelaskan gen awal yg penting kepada jangkitan FIPV dan pertahanan

perumah. Dalam kajian ini, sampel RNA sel ginjal kucing Crandell Rees

(CRFK) yang dijangkiti FIPV serotype II strain 79-1146 selama 3 jam dijujuk

menggunakan platform penjujuk generasi terkini, Illumina™ kemudian

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dianalisis dengan perisian CLC bio Genomic Workbench. Bacaan penjujukan

dipetakan kepada genom rujukan ‘shotgun’ Felis catus 2X beranotasi dan

analisis ekspresi sel kawalan melawan sel yang dijangkiti dijalankan. Ujian

Kal's Z perkadaran ekspresi digunakan untuk menentukan gen yang

diekspresikan secara signifikan. Gen yang dinyatakan dengan kadar

penemuan palsu (FDR) kurang daripada 0.05 dan lebih daripada 1.99

perubahan kali ganda telah dipertimbangkan untuk analisis lanjut. Analisis

RNA-seq memetakan bacaan sel kawalan dan sel yang dijangkiti kepada

18899 gen daripada 19046 gen beranotasi, manakala analisis ekspresi gen

mendedahkan 61 gen berbeza kawal atur oleh kedua-dua sampel dengan 44

gen dinaik kawal atur manakala selebihnya diturun kawal atur. Di antara

gen tersebut adalah kimokin untuk menarik monosit, CCL8 yang diekspresi

hanya di dalam sampel yang dijangkiti mencadangkan bahawa tindak balas

awal terhadap FIPV melibatkan sel imun berantara (CMI). Di samping itu, 4

gen (CXCL10, PHF11, ATF3, IRF1) yang dikaitkan dengan sitokin rembesan

Th1 juga dinaik kawal atur dalam kajian ini. Sementara itu, gen anti-

apoptotic RNF7 dan gen ribosom RPL39 diekspresi hanya dalam sampel

kawalan menunjukkan bahawa FIPV memulakan apoptosis dan

mengganggu penterjemahan protein sel perumah seawal 3 jam selepas

jangkitan. Selain itu, 9 gen pro-apoptotic (CXCL10, MX1, RSAD2, UBA7,

RNF19B, ESE1, BAK1, CASP7, PD-L1) telah dinaik kawal atur manakala lagi

3 gen anti-apoptotic (c-Kit, CKS2, ID-1) diturun ekspresinya. Peranan

terperinci gen tersebut dan gen berbeza kawal atur yang lain dibincangkan.

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Keupayaan virus untuk menyebabkan apoptosis dalam sel CRFK juga

dianalisis dalam tempoh 48 jam pada 12 rangka masa yang berlainan iaitu 3,

9, 12, 15, 18, 21, 24, 27, 30, 36, 42 dan 48 jam menggunakan aliran sitometri

dan pewarnaan annexin-V FITC. Analisis apoptosis mengesahkan bahawa

sel menjalani apoptosis secara signifikan seawal 18 jam selepas jangkitan dan

apoptosis lewat 30 jam selepas jangkitan. Analisis apoptosis mengesahkan

bahawa virus menyebabkan apoptosis pada sel CRFK 18 jam selepas infeksi.

Penemuan kajian ini dijangka memberi maklumat asas berkenaan

patogenesis jangkitan FIPV. Kajian ini telah berjaya mengenal pasti beberapa

calon gen yang bermungkinan memainkan peranan penting dalam

patogenesis FIPV dan telah memperincikan peristiwa penting kematian sel

berikutan jangkitan FIPV dalam sel-sel CRFK.

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ACKNOWLEDGEMENTS

First and foremost, thank you Allah for everything.

I am very grateful to Prof. Dr. Abdul Rahman Omar for being an awesome

supervisor, for the support, guidance and assistance throughout the years of

my master’s research. Also, thank you my co-supervisors Assoc. Prof. Dr. Siti

Suri Arshad and Prof. Dr. Mohd Hair Bejo for the great ideas, suggestions

and assistance.

I would like to thank Assoc. Prof. Dr. Noorjahan Banu Alitheen for giving me

the permission to join her flow cytometry training and to use the flow

cytometry machine at her lab. Thousands thanks to Assoc. Prof. Dr. Goh

Yong Meng and Dr. Yeap Swee Keong for their guidance on apoptosis result

analysis especially for the tricky statistical analysis part.

Special thanks to Virology Lab, Faculty of Veterinary Medicine staff Assoc.

Prof. Dr. Zenathul Nazariah Allaudin, En. Mohd Kamaruddin Awang Isa

and Mrs Siti Khatijah Mohamed. Also to Laboratory of Vaccines and

Immunotherapeutics, Institute of Bioscience staff Mrs Nancy Liew Woan

Cham, Mrs Norhazalina Md. Isa and Mrs Norhafiza Azwa Ghozali.

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My deepest gratitude goes to my parents and my family for the everlasting

support, love and patience. Many thanks go to my close friends for the

encouragement and advice.

Last but not least, thank you to Advanced Medical and Dental Institute

(AMDI), University Sains Malaysia (USM) for the scholarship especially to

Dr. Haji Ramli Saad, Assoc. Prof. Dr. Tang Thean Hock and Mrs. Fatimah

binti Othman.

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I certify that an Examination Committee has met on 14 August 2012 to conduct the final examination of Mohammad Syamsul Reza Bin Harun on his Master of Science thesis entitled "Transcriptome and Apoptosis Analysis of Feline Infectious Peritonitis Virus Infected Crandell Rees Feline Kidney

cells" 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 Master of Science. Members of the Examination Committee were as follows: SHARIDA BINTI FAKURAZI, PhD

Associate Professor Department of Human Anotamy Faculty of Medicine & Health Sciences Universiti Putra Malaysia (Chairman) ZEENATUL NAZARIAH BINTI ALAUDDIN, PhD

Associate Professor Department of Veterinary Pathology & Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) SUHAIMI NAPIS, PhD

Associate Professor Department of Cell and Molecular Biology Faculty of Biotechnology and Science Molecule Universiti Putra Malaysia (Internal Examiner) PETER J.M. ROTTIER, PhD

Professor Department of Infectious Diseases and Immunology Utrecht Univeristy The Netherland (External Examiner)

___________________________________ SEOW HENG FONG, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

x

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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:

Abdul Rahman bin Omar, PhD

Professor

Institute of Bioscience

Universiti Putra Malaysia

(Chairman)

Siti Suri Binti Arshad, PhD

Associate Professor

Faculty of Veterinary Medicine


(Member)

Mohd Hair Bin Bejo, PhD

Professor

Faculty of Veterinary Medicine


(Member)

______________________________

Bujang Bin Kim Huat, PhD

Professor and Deputy Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and

citations which have been duly acknowledged. I also declare that it has not

been previously, and is not concurrently, submitted for any other degree at

Universiti Putra Malaysia or at any other institution.

___________________________________________


Date: 14 August 2012

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL SHEETS x

DECLARATION xii

LIST OF TABLES xvii

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xxii

CHAPTER

1 INTRODUCTION 01

2 LITERATURE REVIEW 05

2.1 The Feline Infectious Peritonitis Virus (FIPV) 05

2.1.1 FCoV Classification 05

2.1.2 FECV to FIPV Mutation Theory 07

2.1.3 FIPV Induced Disease 08

2.2 FIPV Internalization and Replication 08

2.2.1 FIPV Receptors 10

2.3 Prevention, Vaccination and Treatment 11

2.4 FIPV 79-1146 Virus 14

2.5 Cat as Models for Human Viral Disease Studies 19

2.6 Cat Genome 20

2.7 Transcriptome 22

2.7.1 Transcriptome Studies – from Past to Present 23

2.7.2 Next Generation Sequencing (NGS) 24

2.7.3 Illumina Sequencing 25

2.8 Gene Expression Studies of Virus Infected Feline

Cells 28

2.8.1 Feline Immunodeficiency Virus (FIV) in

Lymphocytes and CRFK cells 28

2.8.2 Transcriptome Studies of Low Pathogenic and

Highly Pathogenic FIV Isolates in CRFK Cells 30

2.9 CLC bio™ Genomic Workbench Software 31

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2.10 Apoptosis Associated with FIPV 33

2.10.1 Annexin-V-FITC Flow Cytometry Apoptosis

Analysis 34

3 MATERIALS AND METHODS 36

3.1 Transcriptome Analysis of CRFK Cells Infected

with FIPV 79-1146 36

3.1.1 CRFK Cells Growth 38

3.1.2 Maintaining Cells 38

3.1.3 Cell Subculturing 39

3.1.4 Counting Cells Protocol 40

3.1.5 Virus Propagation 40

3.1.6 Virus Confirmation 42

3.1.7 Virus Titration 43

3.1.8 Control and 3 Hours Infection Samples

Preparation 44

3.1.9 RNA Purification using RNAeasy® Technology 44

3.1.10 Purification of Total RNA from Animal Cells

using Spin Technology 45

3.1.11 Total RNA Quality Analysis 47

3.1.12 CLC bio Genomic Workbench (GWB)

Software Analysis 50

3.1.13 Basic Local Alignment Search Tool (BLAST) 53

3.1.14 Other Bioinformatics Analysis 54

3.2 FIPV 79-1146 Apoptosis Analysis in CRFK Cells 56

3.2.1 CRFK Cells Preparation 56

3.2.2 BD FACSCalibur™ Flow Cytometry Settings

and Statistical Analysis 59

4 RESULTS AND DISCUSSION 61

4.1 Transcriptome Analysis of Early Genes in CRFK Cells

Infected with FIPV 79-1146 61

4.1.1 RT-PCR Result 61

4.1.2 Total RNA Quality and Quantity Analysis 62

4.1.3 CLC bio Results – Sequences Trimming Results 64

4.1.4 CLC bio Results – Expression Analysis Result 66

4.1.4.1 Box Plot 66

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4.1.4.2 Scatter Plot 69

4.1.4.3 Volcano Plot 73

4.1.5 Filtered Genes – Up Regulated Genes BLAST

Results 75

4.1.6 Filtered Genes – Down Regulated Genes

BLAST Results 83

4.2 Discussion - Up Regulated Genes 86

4.2.1 Cytokines/Chemokines 91

4.2.2 Interferon Stimulated Genes (ISG) 94

4.2.3 Transporters 103

4.2.4 Intracellular Defense:Restriction Factor 104

4.2.5 Ubiquitination:E1,E2 and/or E3 108

4.2.6 Cell Surface Receptor 111

4.2.7 Trancription/Co-transcription Regulator 111

4.2.8 Cell Growth/Adhesison/Signaling

Regulators 116

4.2.9 Apoptosis Related Genes 121

4.2.10 Unclassified Genes 124

4.3 Discussion – Down Regulated Genes 126

4.4 General Discussion on Transcriptome Study 139

4.5 PANTHER GO Results 141

4.5.1 Up Regulated Genes:PANTHER Pathway 142

4.5.2 Up Regulated Genes:Biological Process 146

4.5.3 Up Regulated Genes:Molecular Function 150

4.5.4 Up Regulated Genes:Cellular Component 154

4.5.5 Down Regulated Genes:PANTHER Pathway 156

4.5.6 Down Regulated Genes:Biological Process 158

4.5.7 Down Regulated Genes:Molecular Function 161

4.5.8 Down Regulated Genes:Cellular Component 164

4.6 FIPV 79-1146 Apoptosis Analysis in CRFK Cells 166

4.7 General Discussion on Apoptosis Study 173

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5 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH 175

BIBLIOGRAPHY 180

APPENDICES 239

BIODATA OF STUDENT 250

LIST OF PUBLICATIONS 251

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