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  • 1. Pak-US Science and Technology Grant Project“HEPATITIS B VIRUS ASSOCIATED HEPATOCELLULAR CARCINOMA IN PAKISTAN”
    Prof. Dr. IshtiaqQadri (NUST, PK)
    Prof. Dr. AleemSiddiqui (UCSD, US)
  • 2. Health Care Relevance of Genotypes, Liver Markers,
    HBV Mutants, RNA Editing Enzymes to HCC in Pakistan
    OBJECTIVES
    LAB FACILITIES
    NUST Center Virology and Immunology
    Brief introduction of Project Manger, Research Officers’ and other personnel in the Center, Ongoing Projects
    Team at NCVI
    Project Layout
    Nationwide sample collection: PIMS (isl), AKUH (khi), Mayo (lhr),
    Hospital Affiliation
    Pakistan Side, US side
    Outcomes
  • 3. 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
    Year 1
    Year 2
    Year 3
    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
  • 4. PROJECT WORKING SCHEME
    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
    Serology for Viral Markers
    HBsAg, Anti HCV, HBeAg/Anti Hbe, Aphafeto protein
    Liver Function tests
    Full Clinical Assessment & General Physical Examination
    Extraction of HBV DNA from pt. serum
    Liver Biopsy
  • 5. PROJECT WORKING SCHEME
    Liver Biopsy
    Extraction of HBV DNA from pt. serum
    Further confirmation by Gel Electrophoresis
    Extraction of HBV DNA
    Measurement of APOBEC3G, APOBEC3F & APOBEC3B mRNA
    PCR Amplification of HBV DNA
    Cycle Sequencing
    Isolation & Cloning of HBx gene from pts with & without HCC
    Cloning in pGEM3 Plasmid
    PCR Amplification of Enhancer II/Core promoter & precore regions of HBV Genome with forward and reverse primers
    Determination of HBV Genotype by EIA with pre S2 Epitope specific monoclonal antibodies
    Sequence comparison at amino acid levels
    DNA Sequencing with fluorescent labeled primers
    Further genotype confirmation by direct sequencing of Enhancer II core promoter & precore / core promoter genomic regions
    Correlation with IFN-alpha & IFN- gamma levels
    Assembling & Tabulation of data for occurrence of HCC in CLD patients
    Eukaryotic expression vector containing HBV Promoter and CMV Heterologous promoter
    Statistical Analysis
    Phylogenetic Analysis
  • 6. HBV Genome Organization
    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
  • 7. HBV can cause HCC in both cirrhotic and non- cirrhotic patients
    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 )
  • 8. HBV PREVALENCE in the World
  • 9. Flashpoint on Epidemiology Of HBV in Pakistan
    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.TropDoct 1997,27:45-6).
  • 10. Hepatitis B Virus and Pakistan
    Normal Liver
    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)
    HBV infected Liver
  • 11. 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
  • 12. NUST CENTER OF VIROLOGY AND IMMUNOLOGY
    RESEARCH FACILITIES and labs at ncvi
    DNA Sequencing lab
    HPLC LAB
    Antiviral lab
    immunology lab
    Flow cytometerLiAB
    hcv LAB
    hepadnaviradae lab
    hpv LAB
    dengue LAB
    Cell culture lab
    ebv LAB
    Tumour virology lab
    Nano biotech lab
    influenza LAB
    Molecular genetics LAB
    Plant virology lab
  • 13. Hepatitis B virus Group
    NUST Centre of Virology & Immunology (NCVI)
    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
    • 14. Identification of precore/basal core promoter/core gene mutants in HBV positive patients
    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
    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
    Research Assistant
    PhD Scholar, NCVI
    Role of APOBEC’S in RNA editing of HBV genome
  • 15. GENOME SEQUENCESTARGETTED IN HBV
  • 16.
  • 17. 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
  • 18. 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
  • 19. 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.
  • 20.
  • 21.
  • 22. THANK YOU