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