Hepatitis C Virus Genotyping in HCV Positive Patients in Sulaimani ...

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Hepatitis C Virus Genotyping in HCV Positive Patients in Sulaimani Governorate

A Thesis Submitted to the Council of College of Science at the University of Sulaimani in Partial Fulfillment of the Requirements For the Degree of Master of Science in Biology (Microbiology)

By Barham Qasim Mahmood B.Sc. Biology (2010), University of Sulaimani

Supervised by Dr. Sahand Kamaludeen Arif Assistant Professor

March 2017

Newroz 2717

‫‏‏‬ ‫يم‏ ‏‬ ِ ‫بسم‏هللا‏ال َّر ْح َم ِن‏ال َّر ِح‬ ‫ ‏‬.‫س ْب َحانَكَ َ‏َل‏ ِع ْل َم‏لَنَا‏إِ ََّل‏ َما‏ َعلَّ ْمتَنَا‏إِنَّكَ‏أَنتَ ‏ا ْل َعلِي ُم‏ا ْل َح ِكي ُم‬ ُ ‫قَالُوا‏‬ ‫) ‏‬23‫سورة‏البقرة (آيت‏‬

In the name of God, The most gracious and merciful They said, ―Glory be to You! We have no knowledge except what You have taught us. It is you who are the Knowledgeable, the Wise.‖ Quran, al-Baqarah 2:32

Supervisor’s certification I certify that the preparation of thesis titled "Hepatitis C Virus genotyping in HCV positive patients in Sulaimani Governorate" accomplished by (Barham Qasim Mahmood), was prepared under my supervision in the college of Science, at the University of Sulaimani, as partial fulfillment of the requirements for the degree of Master of Science in "Microbiology".

Signature: Name: Dr. Sahand K. Arif Title: Assistant Professor Date:

/

/ 2017

In view of the available recommendations, I forward this thesis for debate by the examining committee.

Signature: Name: Dr. Hoshyar Abdullah Azeez Head of Biology Department. Title: Date:

/

/ 2017

Linguistic Evaluation Certification

I hereby certify that this thesis titled "Hepatitis C Virus genotyping in HCV positive patients in Sulaimani Governorate" prepared by (Barham Qasim Mahmood), has been read and checked and after indicating all the grammatical and spelling mistakes; the thesis was given again to the candidate to make the adequate corrections. After the second reading, I found that the candidate corrected the indicated mistakes. Therefore, I certify that this thesis is free from mistakes.

Signature: Name: Arsto Nasir Ahmed Position: English Department, School of Languages, University of Sulaimani Date:

/

/ 2017

Examining Committee Certification We certify that we have read this thesis entitled "Hepatitis C Virus genotyping in HCV positive patients in Sulaimani Governorate" prepared by (Barham Qasim Mahmood), and as Examining Committee, examined the student in its content and in what is connected with it, and in our opinion it meets the basic requirements toward the degree of Master of Science in Biology "Microbiology".

Signature:

Signature:

Name: Bahrouz Mahmood Amin Jaff

Name: Ali Hattem Hussain

Title: Assistant Professor

Title: Assistant Professor

Date: /

Date: /

/ 2017

(Chairman)

/ 2017

(Member)

Signature:

Signature:

Name: Salih Ahmed Hama

Name: Sahand Kamaluldeen Arif

Title: Lecturer

Title: Assistant Professor

Date: /

Date:

/ 2017

(Member)

/

/ 2017

(Supervisor & Member)

Approved by the Dean of the college of Science.

Signature: Name: Dr. Bakhtiar Q. Aziz Title: Professor Date:

/

/ 2017

Dedication

This Thesis is dedicated to o My family, especially my mother and father. o My beloved wife and my dear daughter (Lanwe) o My supervisor and teachers. o Biology Department-University of Sulaimani. o My friends. o All who offered a helping hand.

Barham

Acknowledgments

Thank you my God, for your infinite grace and making everything possible for me by giving me strength to do this work, thank you for giving me the desire and facilitating the ways to carry out my study. I express my deepest gratitude and appreciation to my supervisor Dr. Sahand K. Arif for his endless patient, support, and encouragement throughout the thesis workings. A word of thanks would go to Dr. Ali Hattem Hussain (Shahid Hadi Consultant Clinic) who helped me a lot in carrying out my thesis. I express my sincere thanks to the administrator of college of Medicine who allowed me to work in their research center. I would like to express my special thanks to all of my best friends who helped me to finish this work.

Barham

Abstract To determine the frequency of the hepatitis C virus (HCV) genotypes among 72 previously Real Time - PCR based diagnosed patients in Sulaimani Governorate - Kurdistan region - Iraq, the all blood samples were confirmed as positive for HCV through reverse transcriptase nested polymerase chain reaction (RT-Nested PCR). For genotyping, the Restriction Fragment Length Polymorphism (RFLP) technique was done for 47 samples on a (174 bp) RTNested PCR amplified fragment within the 5´untranslated region (5´UTR) using four specific restriction enzymes (ScrFI, HinfI, BstNI, and BstUI). Out of the 47 samples, the genotypes were distributed as 34 (72.34%) for subtype 1a, 6 (12.77) for genotype 4, 4 (8.51) for genotype 3a, 1 (2.13%) for subtype 1b, 1 (2.13%) for subtype 2a, and 1 (2.13%) for genotype 5. According to comparison with other findings this data concluded that the pattern of the frequency of HCV genotypes in Sulaimani is similar to that of in Turkey, Iran, and most countries of Europe, and North America while it is different from the pattern included Africa and Arab countries.

I

CONTENTS Titles

Page

Abstract

…………………………………………………………… I

Contents

…………………………………………………………… II

List of Tables

……………………………………………………. VI

List of Figures

……………………………………………………. IX …………………………………………….. XI

List of Abbreviations

Chapter One: Introduction 1. Introduction

…………………………………………………… 1 Chapter Two: Literature Review

2. Literature Review

……………………………………………

2.1. Hepatitis C Virus

………………………………………….... 3

3

2.1.1. Description …………………………………………………... 3 2.1.2. Genomic organization

…………………………………….

3

2.1.3. Life cycle of HCV ………………………………………….... 5 2.2. HCV pathogenesis

………………………………………….... 8

2.3. Transmission …………………………………………………... 9 ……………………………...

11

2.5. Clinical significance of HCV genotypes ……………………....

15

2.4. HCV geographical distribution

2.5.1. Effect of HCV heterogeneity on the treatment ……………….. 15 2.6. Detection of HCV infection ……. ……………………………… 16 2.6.1. Methods for detecting Anti-HCV……………………………... 16 2.6.1.1 First ELISA generation for HCV detection …………………. 17 2.6.1.2. Second ELISA generation for HCV detection ……………… 17

II

2.6.1.3. Third ELISA generation for HCV detection ………………... 18 2.6.1.4. Supplemental tests for anti-HCV …………………………… 18 2.6.2. Methods for detecting HCV RNA……………………………... 19 2.6.3. Genotyping methods

…………………………………….

20

2.6.3.1. Direct sequencing…………………………………………… 20 2.6.3.2. RFLP

…………………………………………………... 21

2.6.3.3. Genotype specific primer

……………………………...

22

2.6.3.4. Hybridization of genotype specific probes ……………….

23

2.6.3.5. Genotype-specific antibodies ……………………………...

23

2.7. Treatment of HCV ……………………………………………

24

2.7.1. Interferon and Ribavirin ………............................................

24

2.7.2. Other new drugs

……………………………………………

24

Chapter Three: Materials and Methods 3.1. Materials

…………………………………………………... 26

3.1.1. Chemicals

……………………………………………

3.1.2. Apparatus

…………………………………………………... 27

26

3.1.3. Kits and contents ……………………………………………. 28 3.1.3.1. STRP™ Hepatitis C Virus Detection Kit …………………… 28 3.1.3.2. Restriction endonucleases …………………………………… 29 3.1.3.3. AccuPower® RocketScript RT-PCR PreMix ………………. 29 3.1.3.4. Viral Nucleic Acid Extraction Kit ΙΙ ………………………... 29 3.1.3.5. OneTwin 100 ……………………………………………..

29

3.2. Methodology …………………………………………………... 30 3.2.1. Specimen collection and storage ……………………………... 30 3.2.2. Detection of HCV ………………………………………...….. 30 III

3.2.2.1. RNA extraction ……………………………………………

30

3.2.2.2. Single tube cDNA Synthesis and first PCR Round PCR …… 31 3.2.2.3. Second round PCR …………………………………………. 31 3.2.3. HCV 5´UTR amplification by RT nested PCR ………………. 32 3.2.3.1. PCR primer design

…………….………………………. 32

3.2.3.2. Preparation of primers stock solution ……………….…..…. 33 3.2.3.3. Viral nucleic acid extraction ……………………...……..…. 33 3.2.3.4. RNA quality and quantity measurement …………………… 34 3.2.3.5. cDNA synthesis ……………………………………………. 34 3.2.3.6. First round …………………….............................................. 34 3.2.3.7. Second round ………………………………..……………... 35 3.2.3.8. Gel documentation for detection of nested PCR bands ..…..

36

3.2.4. Genotyping by RFLP technique ………..……………………

37

3.2.4.1. Gel documentation for detection of RFLP bands ..................

37

3.2.5. HCV 5´UTR sequencing ……………………………...……..

38

Chapter Four: Results 4. Results ………………………………………………………......

39

4.1 HCV detection …………………………………………………

39

4.2. Amplification of HCV 5´UTR …..…………………………....

44

4.3. Genotyping through RFLP method …………………………...

48

4.4. HCV 5´UTR Sequencing ……………………………...............

56

Chapter Five: Discussion 5. Discussion ………………………………………………………

57

Chapter Six: Conclusions and recommendations 6. Conclusions and recommendations ………………………………. 61 IV

Conclusions …………………………………………………………. 61 Recommendations …………………………………………………... 62 Appendix ……………………………………………………………. 63 References …………………………………………………………. 66

V

List of Tables Table No. 2.1

The Title

Page No.

Studies on HCV genotyping in Iraq including Kurdistan region ………………………....

14

3.1

Chemicals ………………………………………………...

26

3.2

Laboratory instruments …………………………………….

27

3.3

The Kits …………………………………………..………..

28

3.4

STRP™ Hepatitis C Virus Detection Kit contents and quantity……………………......

28

3.5

Restriction endonucleases and their sources ….…………..

29

3.6

Viral Nucleic Acid Extraction Kit ΙΙ contents …..…………

29

3.7

OneTwin 100 contents ……..……………………..............

29

3.8.

Nested PCR primers ………………………………………

32

3.9

First and second round PCR reaction components …….......

35

3.10. Nested PCR condition ……………………………………..

36

3.11. RFLP pattern of HCV genotypes adopted from Pohjanpelto, Lappalainen et al. ……………. 4.1

Percentage HCV genotypes among 47 HCV patients in Sulaimani Governorate . …………...……

4.2

38

48

Comparison between RFLP method and sequence analysis of HCV 5´UTR ………………….……..

VI

59

List of Figures Figure No.

The Title

Page No.

2.1

HCV genes and gene products ……………………………

4

2.2

Cellular entry of HCV particles …………………….…….

5

2.3

The infectious viral particle model of replication and assembly ……………………….………...

7

2.4

Natural history of HCV …………………………………..

9

2.5

World prevalence of HCV genotypes ……..……………...

12

2.6.

World HCV prevalence 2012 ……………………..……...

13

4.1a Gel electrophoresis of RT nested PCR amplicons for sample 1-20 ……………………….……....

40

4.1b Gel electrophoresis of RT nested PCR amplicons for sample 21-40 ……………………..……….

41

4.1c Gel electrophoresis of RT nested PCR amplicons for sample 41-60 ………………………………

42

4.1d Gel electrophoresis of RT nested PCR amplicons for sample 61-72 ………………………………

43

4.2a, 4.2b, 4.2c, 4.2d Gel electrophoresis results of

4.3

RT nested PCR for amplification 5´UTR ………….……...

44-47

HCV genotype distributions in Sulaimani Governorate …..

49

IX

4.4a, 4.4b, 4.4c, 4.4d, 4.4e, 4.4f, 4.4g Gel electrophoresis results of RFLP for HCV genotypes ………………………

X

49-55

List of Abbreviations Abbreviations

Meaning

µl

microliter

ALT

Alanine aminotransferase

bp

base pair

bDNA

branched DNA

C

Celsius

CD

Cluster of differentiation

cDNA

complementary deoxyribonucleic acid

CLDN

Claudin

DEPC

Diethylpyrocarbonate

DNA

deoxyribonucleic acid

dNTPs

Deoxynucleotide Triphosphates

DW

Distilled Water

E

Envelope

EDTA

Ethylenediaminetetraacetic acid

EIA

Enzyme immunoassay

ELISA

Enzyme-linked immunosorbent assay

ER

Endoplasmic reticulum

FDA

Food and Drug Administration

g

gram

GTP

Guanosine triphosphate

HAI

Hospital-acquired infection

HCC

Hepatocellular carcinoma

HCV

Hepatitis C Virus XI

IRES

Internal ribosomal entry site

IU

International unit

IVDU

Intravenous drug users

LDL

Low-density lipoproteins

ml

milliliter

NANBH

Non-A non-B hepatitis

NCBI

National Center for Biotechnology Information

NHANES

National Health and Nutrition Examination Survey

NS

Non-structural

NTPase

Nucleotide tri-phosphatase

ORF

Open reading frame

PCR

Polymerase chain reaction

PEG-INF

Pegylated interferons

pmole

picomole

qPCR

Quantitative polymerase chain reaction

RdRp

RNA-dependent RNA polymerase

RFLP

Restriction Fragment Length Polymorphism

RIBA

Recombinant ImmunoBlot Assay

RNA

Ribonucleic acid

RT-Nested PCR

Reverse-transcriptase nested polymerase chain reaction

SIA

Strip Immunoblot Assay

SVR

Sustained Virologic Response

TBE

Tris-borate-EDTA

UTR

Untranslated region

VLDL

Very-low-density lipoproteins XII

Chapter One Introduction

Chapter One

Introduction

1. Introduction

Hepatitis C virus (HCV) infection is an important public health problem, it causes liver disease and is the leading indication for liver transplantation worldwide. More than 180 million people have suffered from this virus and most of them are at risk of developing complications [1]. HCV is an enveloped virus of the Flaviviridae family having seven known genotypes and many subtypes [2]. The viral genome is a single-stranded, positive-sense ribonucleic acid (RNA) molecule with an approximately 9.6 kb containing a single open reading frame coding for all the structural and nonstructural proteins of the virus [3]. There are extensive genetic diversities reported for HCV genomes obtained worldwide, depending on this, HCV sequences obtained from clinical samples have grouped into different genotypes and subtypes [4]. Acute HCV infection is mostly asymptomatic and consists approximately 70% of all cases. About 80% of the infected patients will suffer from the chronic infection and are at high risk for the end stage of liver cirrhosis and hepatocellular carcinoma (HCC). HCC is known as a common cancer worldwide and accounts for about 5.6 % of all types of cancers. It ranks fifth cancer in the world, and the third common cancer caused deaths [5]. HCV shows a remarkably high degree of genetic heterogeneity. There are seven known genotypes (1-7). Different genotypes of HCV have 30-35% diversity at their nucleotide sequences. The genotypes are further divided into subtypes with