A Molecular Approach for Genotyping of Hepatitis B Virus Using Restriction Pattern Analysis of S Amplicon in Pakistan

Abstract

Background Hepatitis B virus (HBV) is the major etiological agent causing acute and chronic liver disease worldwide with significant morbidity and mortality. The high genetic variability of HBV is reflected by eight genotypes (A to H), each with a particular geographical prevalence. The global pattern of HBV genotypes is associated with the distribution of human population among the different continents and reflects the patterns of human migrations. Objectives This study was conducted with following objectives: 1) To study the prevalence of HBV genotype in Pakistani population; 2) To assess that the RFLP system is simple, rapid and standardized way of identifying HBV genotype. Study Design & Method In cross-sectional study design a total of 255 HBV ELISA positive samples were studied in order to identify the most prevalent genotypes in Pakistan. These HBV related patients visited various hospitals in Pakistan at Faisalabad, Lahore and Islamabad. Among these samples, 214 were PCR positive and rest 41 were PCR negative for HBV. S-gene of HBV PCR positive samples was amplified by regular (first round) and nested PCR (second round). Second-round PCR products were digested by Restriction Fragment Length Polymorphism (RFLP). This was carried out using five restriction enzymes (HphI, NciI, AlwI, EarI and NlaIV) that identified the genotype-specific sequences. Results & Conclusion Among (214) PCR positive samples only genotype C and D were identified in local population with 21 cases (9.81%) of genotype C and 195 (91.1%) of genotype D. Hence, the algorithm adopted in this study can be used to identify various HBV genotypes.