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

14. Identification of precore/basal core promoter/core gene mutants in HBV positive patientsResearch 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

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.

22. THANK YOU