Abstract
Hepatitis B Virus (HBV) infection is one of the major causes of liver cirrhosis, hepatocellular carcinoma and deaths due to the acute or chronic consequences worldwide. HBV is distributed into various genotypes based on nucleic acid sequence variation. To develop a method of HBV genotyping and drug resistance interpretation using partial sequencing of polymerase gene. This study was performed on 98 HBV infected patients' serum samples from Western India. A nested PCR protocol was designed for amplification of pol gene from HBV genome and Sanger's sequencing of the gene fragment. Sequences were aligned with HBV reference sequences for phylogenetic analysis and for characterization of genetic diversity. Drug resistance mutations were screened using HBVSeq program from Stanford University. Distribution of HBV genotypes showed predominance of genotype D, circulating in 76 (77.55%) patients (p < 0.05). Genotypes A and C were less prevalent and were identified in 4 (4.08%) and 18 (18.37%) patients, respectively. Anti-retroviral drug resistance mutations were not detected in any patient. A method for determination of HBV genotypes using pol gene sequencing which simultaneously detects major drug resistance mutations has been established. HBV genetic diversity may play an important role in treatment decision.
Highlights
Hepatitis B Virus (HBV) infection is a major health care problem with up to 400 million affected persons worldwide and accounts for one million deaths worldwide from cirrhosis, liver failure African@ 2017 Chavan et al; licensee African Health Sciences
PCR amplification of HBV pol gene Hepatitis B Virus DNA isolated from patients generated 409 bp PCR amplification product for pol gene using nested PCR approach (Figure 1)
This study demonstrated that using HBV DNA polymerase gene sequence all the different genotypes show distinct clades in phylogenetic analysis and the gene fragment used for analysis is a suitable target for HBV genotyping
Summary
HBV infection is a major health care problem with up to 400 million affected persons worldwide and accounts for one million deaths worldwide from cirrhosis, liver failure African@ 2017 Chavan et al; licensee African Health Sciences. Ying et al, performed semi-nested PCR amplification (using three primers) of pol gene and its sequencing for HBV genotyping in Chinese patients.[10] In this study, we used nested PCR that uses four primers that results in increased specificity. As this assay is performed on low viral load samples, it underlines higher sensitivity of nested PCR approach. Conclusion: A method for determination of HBV genotypes using pol gene sequencing which simultaneously detects major drug resistance mutations has been established.
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