Journal of Bacteriology & Parasitology

Abstract

HBV Genotyping and Phylogenetic Analysis through Multiplex PCR Using Type Specific Primer Pairs in Pakistani Population

Javed Iqbal, Abida Raza and Jabar Zaman Khan Khattak

Background: Hepatitis B virus causing acute and chronic hepatocellular carcinoma relics a serious health issue affecting more than 350 million people worldwide. Geographically, nine genotypes of HBV have been identified so far. Four open reading frames named (ORF P, S, F and X) renders it to specifically targets hepatocyte cells. Additionally, reverse transcription process present in the life cycle of HBV caused four order higher mutation rates as compared to adenovirus. The structural and functional differences between HBV genotypes are the mainstay to severity, complications, treatment and possibly vaccination against the virus. Hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, but the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis and medical therapies. In Pakistan, there is no reported data available on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of dominant strains prevalent here.

Objectives: This study was aimed to figure out the prevalence of HBV genotypes among population in Pakistan. In this research, we focused to find out the new strains and sub genotypes of HBV. Pakistan is one of the HBV endemic countries but there is no data on subgenotypes and their recombination or the phylogenetic relatedness of the virus endemic in the country. The present study was carried out in this perspective. This study will help future researcher to study some other aspects of HBV related strains in Pakistan and also about its medical therapies.

Place and duration of study: The present study was carried out at Polymerase Chain Reaction (PCR) lab, Nuclear Medicine, Oncology and Radiotherapy Institute [NORI], Islamabad, and the samples were collected between September 2012 to February 2014.

Study design: A total of 450 Hepatitis B surface antigen HBsAg and HBV DNA positive samples were collected in the first step during the period of September 2012 to February 2014 and sent to Nuclear Medicine Oncology and Radiotherapy Institute [NORI], for further analysis. All the samples were genotyped by type specific nested PCR primer pair method for 8 HBV genotypes from A through H. The patients were randomly selected irrespective of their age and gender and a written consent (parental consent in case of less than 18 years of age) was obtained. The study was approved by ethical review committee. In our research, we have used the type specific primer technique to find out the dominant strain in Pakistani populations and furthermore its phylogenetic analysis with reference sequences retrieved from NCBI databases.

Results: Our results showed a direct relationship between viral load and identified genotypes and pinpointed that genotype D is the most pre-dominant genotype and prevailing in high proportions among all genotypes in Pakistani population. Mix HBV genotype infections of genotype A with B & C, D with B and C constitute about 17 percent of all the samples. Subgenotypes of different HBV strains have been identified on the basis of differences in the complete nucleotide sequences. Positive PCR results were repeated twice for confirmation. Furthermore, phylogenetic analysis revealed a homology between our reported sequences and reference sequences. The phylogenetic method showed that genotypes/sub-genotypes varies in the geographical areas and correlate strongly with ethnicity.

Conclusion: Genotype D was identified and come out to be the most dominant genotype in Pakistani community showing sub-genotypes of D1 & D3 in our study. Genotype A and D are present as co-infection with each other and contributed as the second prevailing genotypic group. Additionally, phylogenetic analysis revealed that genotypes varies in the geographical areas and correlate strongly with reference sequences from NCBI.