Sabihur Rahman Farooqui, Masarrat Afroz, Syed Ali Azam, Siddiqui, Syed Naqui Kazim
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi -110025, India.

ABSTRACT

The potential for hepatitis B virus (HBV) to alter its genome is considerable. This occurs because the virus utilizes a reverse transcription step in replicating the viral genome. HBV mutants with survival advantages over the wild type virus appear within the selective in vivo environment. Some of these viruses have escape, antiviral resistant mutantions in its surface and overlapping polymerase and others may have mutations in the precore region or in the basal core promoter region. The most significant and commonly occurring mutation in the precore region is G1896A (W28Stop) which results into premature truncation of Hepatitis B virus precore gene and eventually inhibits the HBeAg secretion in the blood of patients. This event is manifested as HBeAg negative serology despite the presence of active viral replication among chronically infected HBV patients, commonly designated as e negative chronic hepatitis B. Virus is known to adapt this strategy generally in order to evade the immunological clearance. Therefore, this stop codon mutation is also known as immune pressure mutation. On the other hand majority of available literatures describes the absence of this stop codon mutation among patients showing HBeAg positive serology. Unlike the findings of earlier available literatures here we report the presence of W28Stop codon mutation among HBeAg positive chronic hepatitis B patients. From the thirty HBeAg positive chronic hepatitis B patients viral DNA was extracted with the help of standard phenol:chloroform:isoamyl alcohol method from the sera samples. Nested PCR was performed using the extracted viral DNA which resulted in the generation of 320 bp (from 1653 to 1972) amplicon. PCR products were purified by the commercially available PCR purification kit. Purified PCR products were subsequently sequenced with respective forward and reverse primers. The sequences of viral DNA spanning nucleotide 1701 to 1930 of viral genome were aligned with that of known genotypes i.e. A-G with the help of Multalin sequence alignment programme. Thereafter the respective DNA sequences obtained from patients samples as well as from the standard genotypes (A-G) were converted into corresponding amino acids sequences with the help of Expasy multipurpose bioinformatics software. Amino acid sequence alignment was subsequently performed. Five of thirty studied patients interestingly showed the presence of classic G1896A (W28Stop) codon mutation in their precore gene. This unusual finding could possibly be due to the presence of mixed viral population of wildtype and mutated viral genome. Secondly, the studied samples could have coincided with the time point of that window period from the patient where the HBeAg positive patients generally tend to clear the viral infection (seroconversion). At this time point generally virus encounters a significantly higher immune pressure. Further to this an interesting observation in this study is that all the five patients having W28Stop mutation showed homology with HBV genotype A. Read more…