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