Zaheenul Islam Siddiqui, Syed Ali Azam, Masarrat Afroz, Sabihur Rahman Farooqui, Syed Naqui Kazim
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi -11002, India.
Hepatitis B virus (HBV) is a member of Hepadnaviridae family. Most cases (around 80%) of hepatocellular carcinoma (HCC) in India are due to chronic infection with HBV and/or Hepatitis C virus (HCV). HBV can be grouped in eight genotypes A-H with more than 8% DNA sequence divergence between the groups. There is still lack of sufficient data regarding the HBV genotypes based on the whole genome sequence. Therefore we aimed to carry out the present study on HBV genotypes based on analysis of nearly three fourth of the whole genome which also contributes to the major and clinically important regions within the viral genome. Viral DNA was extracted from sera samples of fourteen patients, with the help of standard phenol/chloroform extraction method. The obtained viral DNA was used as a template for nested PCR amplification of three regions- Pre-S1/2 region, part of surface and overlapping polymerase region and precore/core region of HBV genome. For this, different primers specific for these regions were used. Purified PCR products were bidirectionally sequenced with respective primers in an automated DNA sequencer. Obtained sequences were translated to amino acid sequences using the ExPasy bioinformatics tool. DNA sequences of Genotypes A-G were downloaded from Gene bank according to the published accession numbers for the respective genotypes, and translated to amino acid sequences with the help of ExPasy tool. For each isolate, amino acid sequences of the mentioned three regions were aligned with amino acid sequences of the respective regions of genotypes A-G with the help of Multalin alignment programme. After carefully analyzing the data we were able to categorize these fourteen studied patients into three categories of genotypes: 1. Seven out of eleven patients showed complete homology in all the three regions of viral genome with genotype A with consistent presence of 3-5 loci of variations. Interestingly these variations were in sole agreement with genotype D. 2. Four patients had complete sequence homology with genotype D with consistent sequence variations at 3-5 loci. Again, these variations were in agreement with genotype A. 3. Three patients showed a unique recombination of the two genotypes, i.e. A and D. In these three patients, whole pre-S1 and pre-S2 regions had homology with genotype A except at some loci as observed in the first category. On the other hand, surface/polymerase and precore/core regions have homology with genotype D except some sequence variations at specific loci as observed in the second category. None of the fourteen patients had hundred percent homology with respect to their amino acids, exclusively with the sequences of any one of the known genotypes. To conclude, Indian patients infected with HBV harbor either genotype A or D in presence of consistent occurrence of amino acid sequence variations at specific loci. The obtained data in the present study could also be a possible evidence of existence of recombinant of A and D genotypes of HBV in few patients. However the evidence of recombination in this study needs further elaborated study taking the account of clones of whole genome sequences from patients’ isolates. Read more…