Abstract
BackgroundAs the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis. In Pakistan, there is no reported data on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of mutations present in the strains prevalent here.Findingsto confirm specificity of PCR typing, phylogenetic analysis of the pre-S1 region and the divergence was studied through 13 sequences of 362 bp (accession number EF432765 – EF432777). A total of 315 serum samples, selected from HBsAg positive patients representing the major ethnic groups, residing in Karachi, Sindh were tested for genotyping. Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients. The rest of the genotypes A and a mixture of A and D (AD) were distributed as 20%, and 10% respectively. Phylogenetic tree demonstrated clustering of 11 samples with subgenotype D1 sequences and the remaining two strains on a branch within D3 samples. All samples intermixed with strains from other countries and were found to be closely related to Indian, Iranian and Egyptian HBV strains with 98.7 – 99.0% homology.ConclusionThis study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients. More studies are required to investigate the reason for fewer inclusions of D3 compared to the D1 in Pakistani HBV strains.
Highlights
As the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis
This study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients
Prevalence of HBV/D as the most common genotype Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients
Summary
Specimens Blood was drawn from 350 patients undergoing treatment at Pakistan Medical Research Council (PMRC) and Ziauddin University Hospital (ZUH), diagnosed positive for HBV by HBsAg (ELISA – MUREX kit by Abbott Laboratories). The step one PCR was carried out with universal outer primers P1 (sense) and S12 (antisense) in a tube containing 50 μl of a reaction buffer made up of the following components: 50 ng of each outer primer, a 200 μM concentration of each of the four deoxynucleotides, 1 U of Taq polymerase (Perkin-Elmer, Norwalk, Conn.), and 10× PCR buffer containing 1.5 mM MgCl2. A 2.5 μl aliquot of the first PCR product was added to two tubes containing the second sets of each of the inner primer pairs, each of the deoxynucleotides, Taq polymerase and PCR buffer, as in the first reaction and amplified for 35 cycles with: preheating at 94°C for 3 min, 15 cycles of amplification at 94°C for 20 s, 58°C for 30 s, and 72°C for 40 s, and an additional 20 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s with an extension of 7 min.
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