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.
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N. Badar, U. Farooq, S. Ali, N. Nisar, M. Abubakar and J. Qureshi, "A Molecular Approach for Genotyping of Hepatitis B Virus Using Restriction Pattern Analysis of S Amplicon in Pakistan," Open Journal of Medical Microbiology, Vol. 2 No. 1, 2012, pp. 16-23. doi: 10.4236/ojmm.2012.21003.
 C. Hannoun, P. Horal and M. Lindh, “Long Term Mutation Rates in the Hepatitis B Virus Genome,” Journal of General Virology, Vol. 81, No. 1, 2000, pp. 75-83.
 N. Khanna, D. Sahal, S. Swaminathan, K. R. Baba, E. Alalan, A. Chauhan, D.A. Chugh, M. Malhotro and J. Singh, “Mammalian Biology: Recombinant Gene Product,” International Centre for Genetic Engineering and Biotechnology, 1997.
 J. M. Francis, “Updates on Diagnosis, Management and Prevention of Hepatitis B Virus Infection,” Journal of Clinical Microbiology, Vol. 12, No. 2, 1999, pp. 355-366.
 V. A. Mahoney, J. L. Lopez, P. F. Talenta, G. Palacios, I. Badia, A. Ferro, C. Galoppo and R. Campos, “Distribution of Hepatitis B Virus Genotypes in Two Different Pediatric Populations from Argentina,” Journal of Clinical Microbiology, Vol. 36, No. 11, 1999, pp. 3362-3365.
 V. A. Mahoney, J. Francis and K. Mark, “Vaccines,” 3rd Edition, Orenstein editors Company, Philadelphia, 1999.
 M. Lindh, A. S. Andersson and A. Gusdal, “Genotypes nt, 1858 Variants and Geographic Origion of Hepatitis B Virus, Large Scale Analysis Using a New Genotyping Method,” Journal of Infectious Diseases, Vol. 175, No. 6, 1997, pp. 1285-1293. doi:10.1086/516458
 M. Lindh, P. Horal, A. P. Dhillon and G. Norkans, “Core Promoter Mutation and Genotypes in Relation to Viral Replication and Liver Damage in East Asian Hepatitis B Virus Carriers,” Journal of Infectious Diseases, Vol. 179, No. 4, 1999, pp. 775-782. doi:10.1086/314688
 World Health Organization, Int. Agn. For Research on Cancer Monograph, Vol. 59, 1994, p. 45.
 S. J. Zuberi, “An Overview of HBV/HCV in Pakistan,” Journal of the Pakistan Medical Association, Vol. 37, 1998, pp. 12-17.
 B. Weber, W. Melchior, R. Gehrke, H. W. Doerr, A. Berger and H. Rabenau, “Hepatitis B Virus Markers in Anti HBc Only Positive Individuals,” Journal of Medical Virology, Vol. 64, No. 3, 2001, pp. 312-319.
 M. Mizokammi, T. Nakano, E. Orito, Y. Tanaka, H. Sakugawa, M. Mukaide and B. H. Robertson, “Hepatitis B Virus Genotype Assignment Using Restriction Fragment Length Polymorphism Patterns,” FEBS Letters, Vol. 450, No. 1, 1999, pp. 66-71.
 S. M. Bower and G. M. Sim, “Relationship within and between the Genotypes of Hepatitis B Virus at Point across the Genome: Footprints of Recombination in Certain Isolates,” Journal of General Virology, Vol. 81, No. 2, 2000, pp. 379-392.
 J. H. Kao, N. H. Wu, P. J. Chen, M. Y. Lai and D. S. Chen, “Hepatitis B Genotypes and the Response to Interferon Therapy,” Journal of Hepatology, Vol. 33, No. 11, 2000, pp. 998-1002.
 H. Okamoto, F. Tsuda, H. Sakugawa, R. I. Sastrosoewignjo, M. Imai, Y. Miyakawa and M. Mayumi, “Typing Hepatitis B Virus by Homology in Nucleotide Sequence: Comparison of Surface Antigen Subtypes,” Journal of General Virology, Vol. 69, No. 10, 1988, pp. 2575-2583.
 H. Norder, B. Hammas, S. Lofdhal, A. M. Courouce and L. O. Magnius, “Comparison of the Amino acid Sequence of Nine Different Serotypes of Hepatitis B Surface Antigen and Genomic Classification of the Corresponding Hepatitis B Virus Strains,” Journal of General Virology, Vol. 73, No. 5, 1992, pp. 1201-1208.
 L. O. Magnius and H. Norder, “Subtypes, Genotypes and Molecular Epidemiology of the Hepatitis B Virus Reflected by Sequence Variability of S-Gene,” Intervirology, Vol. 38, No. 1-2, 1995, pp. 24-34.
 L. Stuyver, S. De-Gendt, C. Van-Geyt, F. Zoulim, M. Fried, R. F. Schinazi and R. Rossau, “A New Genotype of Hepatitis B Virus: Complete Genome and Phylogenetic Relatedness,” Journal of General Virology, Vol. 81, Pt. 1, 2000, pp. 67-74.
 Fleeming. “The Origion and Evolution of Hepatitis Virus in Humans,” Journal of General Virology, Vol. 82, Pt. 4, 2000, pp. 693-712.
 P. L. Bolyky, A. Rambaut, N. Grassly, W. F. Carman and E. C. Holmes, “Hepatitis B Virus Has a Recent New World Evolutionary Origion,” Hepatology, Vol. 28, 1997, pp. 765-775.
 S. M. Bowyer, L. Van Staden, M. C. Kew and J. G. Sim, “A Unique Segment of the Hepatitis B Virus Group A Genotype Identified in Isolates from South Africa,” Journal of General Virology, Vol. 78, No. 7, 1997, pp. 1719- 1729.
 K. Kidd-Ljunggren, M. Oberg and A. H. Kidd, “The Hepatitis B Virus X Gene: Analysis of Functional domain Variation and Gene Phylogeny Using Multiple Sequences,” Journal of General Virology, Vol. 76, No. 9, 1995, pp. 2119-2130. doi:10.1099/0022-1317-76-9-2119
 C. Van Geyt, S. De-Gendt, A. Rombout, A. Wyseur, G. Maertens, R. Rossau and L. Suyver, “A Line Probe Assay for Hepatitis B Virus Genotypes,” In: R. F. Schinazi, J. P. Sommadossi, H. C. Thomas, Eds, In Therapies for Viral Hepatitis, International Medical press, London, 1998, pp. 139-145.
 M. Hussain, C. Chu, E. Sablon and A. S. F. Lok, “Rapid and Sensitive Assays for Determination of Hepatitis B Virus (HBV) Genotypes and Detection of HBV Precore and Core Promoter Variants,” Journal of Clinical Microbiology, Vol. 41, No. 8, 2003, pp. 3699-3705.
 M. Mizokami, E. Orito, K. Ohba, K. Ikeo, J. Y. N. Lau and T. Gojobori, “Constrained Evolution of Hepatitis B Virus with Overlapping Genes,” Journal of Molecular Evolution, Vol. 44, 1997, pp. 83-90.
 C. Grandjacques, P. Pradat, L. Stuyver, M. Chevallier, P. Chevallier, C. Pichoud, M. Maisonnas, C. Trepo and F. Zoulim, “Rapid Detection of Genotypes and Mutations in the Pre-Core Promoter and the Pre-Core Region of Hepatitis B Virus Genome: Correlation with Viral Persistence and Disease Severity,” Journal of Hepatology, Vol. 33, No. 3, 2000, pp. 430-439.
 H. Naito, S. Havashi and K. Abe, “Rapid and Specific Genotyping System for Hepatitis B Virus Corresponding to Six Major Genotypes by PCR Using Type-Specific Primers,” Journal of Clinical Microbiology, Vol. 39, No. 4, 2001, pp. 1686-1689.
 A. L. Ballard and E. H. Boxall, “Colourimetric Point Mutation Assay: for Detection of Precore Mutants of Hepatitis,” Journal of Virological Methods, Vol. 67, No. 2, 1997, pp. 143-152.
 S. Minamitani, S. Nishiguchi, T. Kuroki, S. Otani and T. Monna, “Detection by Ligase Chain Reaction of Precore Mutant of Hepatitis B Virus,” Hepatology, Vol. 25, No. 1, 1997, pp. 216-222. doi:10.1002/hep.510250139
 S. H. Usuda, H. Okamoto, K. Iwanari, F. Baba, Y. Tsuda and M. Mayumi, “Serological Detection of Hepatitis B Virus Genotypes by ELISA with Monoclonal Antibodies to Type-Specific Epitopes in the PreS2-Region Product,” Journal of Virological Methods, Vol. 80, No. 1, 1999, pp. 97-112. doi:10.1016/S0166-0934(99)00039-7
 J. Sambrook, E. F. Fritsch and T. Maniatis, “Molecular Cloning, a Laboratory Manual,” 2nd Edition, Gold Spring Harbor Laboratory Press, 1989.
 A. M. Courouce-Pauty, J. M. Lemaire and J. F. Roux, “New Hepatitis B Surface Antigen Subtypes inside the Adcategory,” Vox Sanguinis, Vol. 35, No. 5, 1978, pp. 304-308. doi:10.1111/j.1423-0410.1978.tb02939.x
 K. Nishioka, A. G. Levin and M. J. Simons, “Hepatitis B Antigen, Antigen Subtypes and Hepatitis B Antibody in Normal Subjects and Patients with Liver Disease,” Bulletin World Health Organization, Vol. 52, No. 3, 1975, pp. 293-300.
 N. Leung, “Treatment of Chronic Hepatitis B: Case Selection and Duration of Therapy,” Journal of Gastroenterology and Hepatology, Vol. 17, No. 4, 2002, pp. 409-414. doi:10.1046/j.1440-1746.2002.02767.x
 F. Pfeffel, C. Oesterricher, E. Penner, P. Erenci, C. Sillber, R. Fiedler and C. Muller, “Rational Use of Polymerase Chain Reaction Based Detection of Viral Genome in Patients with Serological Marker of Hepatitis B or C Virus Infection,” Wiener Klinische Wochenschrift, Vol. 109, No. 1, 1997, pp. 20-24.
 C. J. Chu, M. Hussain and A. S. Lok, “Hepatitis B Virus Genotype B Is Associated with Earlier HBeAg Seroconversion Compared to Hepatitis B Virus Genotype C,” Gastroenterology, Vol. 122, No. 7, 2002, pp. 1756-1762.
 A. Muqaddas, “Genetic Variation in Well Characterized Chronic HBV Isolates of Pakistan,” Thesis, University of the Punjab, Lahore, 2001.
 X. Ding, M. Mizokami, G. B. Y, et al., “Hepatitis B Virus Genotype Distribution among Chronic Hepatitis B Virus Carriers in Shanghai, China,” Intervirology, Vol. 44, No. 1, 2001, pp. 43-47. doi:10.1159/000050029
 J. H. Kao, P. J. Chen, M. Y. Lai and D. S. Chen, “Genotypes and Clinical Phenotypes of Hepatitis B Virus in Patients with Chronic Hepatitis B Virus Infection,” Journal of Clinical Microbiology, Vol. 40, No. 11, 2002, pp. 1207-1209. doi:10.1128/JCM.40.11.4068-4071.2002
 M. Lindh, P. Horal, A. P. Dhillon and G. Norkans, “Hepatitis B Virus DNA Levels Precore Mutations: Genotypes and Histological Activity in Chronic Hepatitis B,” Journal of Viral Hepatitis, Vol. 7, No. 4, 2000, pp. 258-268.