Pak Us Science And Technology Grant Project E Dited
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Transcript of Pak Us Science And Technology Grant Project E Dited
Pak-US Science and Technology Grant Project
“HEPATITIS B VIRUS ASSOCIATED
HEPATOCELLULAR CARCINOMA IN
PAKISTAN”
Prof. Dr. Ishtiaq Qadri (NUST, PK)
Prof. Dr. Aleem Siddiqui (UCSD, US)
Outcomes
NUST Center Virology and Immunology
Brief introduction of Project Manger, Research Officers’
and other personnel in the Center, Ongoing Projects
•Project Layout
Health Care Relevance of Genotypes, Liver Markers,
HBV Mutants, RNA Editing Enzymes to HCC in Pakistan
Nationwide sample collection: PIMS (isl), AKUH
(khi), Mayo (lhr),
Pakistan Side, US side
OBJECTIVES
LAB FACILITIES
Team at NCVI
Hospital
Affiliation
• To conduct a molecular epidemiological study for characterization of HBV genotypes in Pakistan.
• To identify prevalence of virus specific genetic mutations, which place hepatitis B chronic patients at high risk of liver failure and hepatocellular carcinoma.
• To identity the role of HBV encoded HBx protein and the cellular editing enzymes (APOBEC1-3) in HBV chronicity.
•Objectives
Analysis of PreC/C,
BCP/EnII, X and liver
cytokines levels in
HCC and non HCC
patients
Sample Collection from
HCC and Non HCC patients, Genotyping
and Liver Enzyme levels measurements
Transfections of
cellular editing
enzymes and HBV
proteins in vitro
Year 1 Year 2 Year 3
HOSPITALSPIMS(Isl),
AKUH(Khi),Mayo(Lhr)
Selection of patients of HBV(Chronic carriers) with & without HCC
Written informed consent for participation of study
Patient path towards Hepadna virus lab of NCVI
Full Clinical
Assessment &
General
Physical
Examination
Serology for
Viral MarkersHBsAg, Anti
HCV,
HBeAg/Anti
Hbe, Apha
feto protein
Liver
Function
tests
Extraction of
HBV DNA
from pt.
serum
Liver Biopsy
PROJECT WORKING SCHEME
Extraction of HBV DNA
from pt. serum
Liver
Biopsy
Further confirmation by
Gel Electrophoresis
PCR Amplification of HBV DNA
PCR Amplification of Enhancer
II/Core promoter & precore regions of
HBV Genome with forward and
reverse primers
DNA Sequencing
with fluorescent
labeled primers
Assembling & Tabulation of data for
occurrence of HCC in CLD patients
Statistical Analysis
Cycle Sequencing
Determination of HBV
Genotype by EIA with pre S2
Epitope specific monoclonal
antibodies
Further genotype confirmation by direct
sequencing of Enhancer II core promoter &
precore / core promoter genomic regions
PhylogeneticAnalysis
Extraction of
HBV DNA
Isolation & Cloning of HBx gene from pts with & without HCC
Sequence comparison
at amino acid levels
Measurement of
APOBEC3G,
APOBEC3F &
APOBEC3B mRNA
Correlation with IFN-
alpha & IFN- gamma
levels
Eukaryotic expression vector containing HBV Promoter and CMV
Heterologous promoter
Cloning in
pGEM3
Plasmid
PROJECT WORKING SCHEME
• HBV is a DNA virus, but replicates via reverse transcription of an RNA pregenome.
• The virions contain a partially circular DNA of 3200 nucleotides and encodes for surface antigen (HBsAg), core and e antigens (HBcAg, HBeAg), reverse transcriptase and HBx polypeptide of 152 amino acids (Fig. )
• HBV replication status (viral load) plays an important role in determining the risk of development of HCC. There are eight genotypes of HBV that have distinct geographical distribution.
• Genotype A and D have been shown to be prevalent in Pakistan
HBV Genome Organization
• Our previous work suggests that HBV induces an oxidative stress in infected hepatocytes*,**.
• Elevated ROS activates cellular kinases, which then activates several latent transcription factors such as STAT-3 & NF-kappa B leading to oncogenesis*.
• ROS can also cause DNA damage and if not repaired can lead to genomic instability, high mutation rate and ultimate liver oncogenesis**.
*(Waris et. Al. 2004. Mol. Cell. Biol. 21, 7721-30)**(Qadri et. Al. 2004 Biochem. J. 378, 919–928 )
HBV can cause HCC in both
cirrhotic and non- cirrhotic patients
•HBV PREVALENCE in the World
Geographic Region %
Northern, Western, and Central
Europe, North America, Australia
0.2-0.5 %
Eastern Europe, the
Mediterranean, Russia and the
Russian Federation, Southwest Asia,
Central and South America
2-7 %
Parts of China, Southeast Asia,
tropical Africa
8-20
• Infection with HBV leads to a wide spectrum of clinical
presentations, ranging from asymptomatic carrier state to
acute self-limiting infection or fulminant hepatic failure,
chronic hepatitis with progression to cirrhosis, and
hepatocellular carcinoma (HCC).
• In Pakistan, there are estimated 7-9 million carriers of
hepatitis B virus (HBV) with a carrier rate of 3-5%*.
Predominant genotype D.
• Horizontal transmission, particularly in early childhood,
accounts for most cases of chronic HBV infection in
intermediate prevalence areas like Pakistan **
*(Ali et al, Virology Journal 2011, 8:102)
**(Abdul-Mujeeb S et. Al. Trop Doct 1997,27:45-6).
Flashpoint on Epidemiology Of HBV in Pakistan
• 9 million carriers of hepatitis B
virus (HBV) in Pakistan
• general population 4.33%
• healthy blood donors 3.93%
• military recruits 4.276%
• healthcare persons 3.25%
• pregnant women 5.872%
• prisoners 5.75%
• surgical patients 7.397%
• cirrhosis 28.87%
• HCC 22%
• Genotype D 63.71%
(Ali et al, Virology Journal 2011, 8:102)
Hepatitis B Virus and Pakistan
Some More Facts
• WHO allows 3.5 injections/person/year
• WHO, AKU and PMRC study showed Pakistan has 14
injection/person/year** (Khan AJ: Unsafe injections and the transmission of hepatitis B and C in a Periurban
community in Pakistan. Bull World Health Organ)
Afghan refuges in Pakistan, IDUs, professional blood donors, health care
professionals, prisoners, multiple transfused patients, patients with HCC,
psychiatric patients, general population of some specific areas like Southern
Punjab, Interior Sindh, District,Tatta, Kurrum agency, Baltistan and some
areas of Lahore have very high HBV prevalence of more than 5%, and there
is urgent need of mass vaccination and awareness programs (Ali et al,
Virology Journal 2011, 8:102)
At present there is no study that describes cellular or molecular mechanism
of HBV infection and its progression to HCC in Pakistani population
RESEARCH FACILITIES AND LABS AT NCVI
•NUST CENTER OF VIROLOGY AND IMMUNOLOGY
ANTIVIRAL LAB
NANO BIOTECH LAB
TUMOUR VIROLOGY LAB
CELL CULTURE LAB
HPLC LAB DNA SEQUENCING LAB
FLOW CYTOMETER LIAB
HEPADNAVIRADAE LAB
HCV LAB
DENGUE LAB
INFLUENZA LAB
HPV LAB
MOLECULAR GENETICS LAB
IMMUNOLOGY LAB
EBV LAB
PLANT VIROLOGY LAB
NUST Centre of Virology & Immunology (NCVI)
Hepatitis B virus Group
• Research Officer
• PhD Scholar, NCVI
• Cloning and expression
of S gene in yeast,
Identification of S gene
mutants in Pakistani
population
• Polymerase gene
mutants with reference
to drug resistance
Project Manager
• MBBS Doctor
• PhD Scholar,NCVI
• Managing biopsies, Project Monitoring
• Clinical Supervision,Communications
with clinical Collaborators, Data
Assembling and Tabulation
coordination
Research Officer
• PhD Scholar, NCVI
• Identification of
precore/basal core
promoter/core
gene mutants in
HBV positive
patientsResearch Assistant
• PhD Scholar, NCVI
Role of APOBEC’S in RNA
editing of HBV genome
MPhil Student
• PhD Scholar, NCVI
• Cloning and expression of X gene in
HepG2 cells and it’s association with
cellular factors,Identification of X gene
Mutants in HCC and non HCC patients
Surface gene (Nucleotides155-
831)
Precore (Nucleotides 1814–1900)
Enhancer II (Nucleotides 1685–1773),
Basal Core Promoter
(Nucleotides 1742–1849
complete core region (nt 1901–
2450)
X gene (1374-1838)
Polymerase (2307-3128, 1-
1623)
GENOME SEQUENCES TARGETTED IN HBV
The HBV group has already sequenced the X gene, Precore, core gene, Surface gene and some part of Polymerase
•S Mutants in Pakistan confirmed in
our S gene sequences
• The host's humoral response targets HBV through the hydrophilic
region of the HBsAg between amino acid residues 100 and 160.Thus, mutation(s) in this region would afford HBV variants a
distinct survival advantage
• The common mutations that have been described in this region
include Asp-144-Ala, Met-133-Leu, Gln -129 - His and ILe/Thr -
126 – Ala
• HBsAg or S mutations have now been documented in many
areas of the world but are most common in Asian infants (2% to
3% of vaccine recipients
•S Mutants---continued• Some of the S mutations have been described in
association with other clinical events:
• nucleotide insertions in the area of amino acids 121-124,which can result in false-negative HBsAg testing and
thereby represent a risk to the health of the undiagnosed
patient and the safety of the blood transfusion system
• Despite encouraging results in vaccinated chimpanzees,
HBIg and HBV vaccination do not protect humans from S
mutant infections
•This work needs to be taken to the
other ORF’S of HBV in order to map
their relevance to the Health Care
System in Pakistan.
THANK YOU