Molecular characterization of Hepatitis B virus basal core promoter and precore region of isolates from chronic Hepatitis B patients

Objective: The aim of this study was to analyze mutations in precore/core promoter region of HBV genome in chronic hepatitis B patients from three cities of Pakistan. Methods: A total of 50 treatment naïve chronic hepatitis B patients from Pakistan were selected. Viral load, HBeAg/antiHBe status, HBV ELISA and ALT levels were determined. Direct sequencing of BCP and PC region of HBV genome was carried out following a nested PCR approach. Phylogenetic tree was constructed using MEGA software version 6.0. Statistical analysis was carried out using SPSS version 16.0. Results: The G1896A precore stop codon mutation was detected in 19 (38%) isolates. The mutation was present in 17(34%) isolates from HBeAg negative patients and 2(4%) isolates from HBeAg positive patients. The Classic A1762T/G1764A double mutation was noted in 15 (30%) isolates. Mutation at position 1764 was observed in 12 (48%) samples. A rare G1764T mutation was also detected in 6 (12%) isolates. The CG1802-1803 mutation was detected in 47(94%) isolates. The T1858 mutation was detected in all 50 (100%) isolates. The GCAC Kozak sequence was present in 43(86%) isolates. The CAA1817-1819 mutation was observed in 49(98%) isolates and G1888 mutation was detected in 49(98%) isolates. Overall, 9(18%) isolates had wild-type sequences at all important loci including positions 1762,1764 and 1896. The pattern of sequences at genotype specific positions and phylogenetic tree revealed that majority of study isolates belonged to genotype D. Conclusions: Sequences results showed that precore region was comparatively more conserved than BCP region. Continuous...

Prevalence of Hepatitis B Virus (HBV) Basal Core Promoter/Precore region molecular variants among HIV/HBV co-infected and HBV mono-infected patients in Ile-Ife, Nigeria

IntroductionEvolution of phenotypic diversity among viruses occurs as an escape mechanism against host immune pressure or drug selective pressure. Among HIV/HBV co-infected individuals, various HBV basal core promoter (BCP)/precore (PC) region molecular mutants had been reported with associated phenotypic defect in HBeAg production. The emergence of HBeAg negative variants of HBV in HIV co-infected individuals have profound implication on the diagnosis, management and prognosis of this subset of individuals. This includes delayed clearance of HBV, early development of adverse hepatic events such as liver cirrhosis and hepatocellular carcinoma. Currently, little is known about HBV BCP/PC region genomic heterogeneity in HIV/HBV co-infected patients in Nigeria. Therefore, this study was focussed on investigating evidence of precore/core region genomic variability among HIV/HBV co-infected patients in Nigeria.Materials and methodsA total of 40 patients (20 HIV/HBV co-infected and 20 HBV mono-infected samples) were enrolled into the study and subsequently tested for HBsAg, HBeAg and HBeAb using specific Enzyme-Linked Immunosorbent Assay (ELISA). The BCP/PC genome regions (nucleotides 1653-1959) were amplified using a nested PCR assay and then subjected to BCP/PC mutational analysis in genome sites affecting HBeAg expression especially at the BCP transcriptional and PC Translational stop codon sites.ResultsOverall, 5(83.3%) of the six exploitable sequences after analysis showed various BCP/PC mutations. Only 1(16.6%) sequence from an HIV/HBV co-infected patient had the BCP transcriptional (double mutation; A1762T/G1764A) mutant. Analysis of the PC translational stop codon showed 4 (66.6%) having the G1896A mutants while 33.3% (2) had G1899A mutants.ConclusionThis study has broadened the available evidence of BCP/PC region molecular mutants among HIV/HBV co-infected patients in Nigeria and assessed the difference of mutation prevalence in comparison with HBV mono-infected cohort. We therefore recommend that HIV/HBV co-infected patients be routinely screened for hepatitis B virus precore region mutants to improve their patient outcome.

Virological Factors Associated with the Occurrence of HBV Reactivation in Patients with Resolved HBV Infection Analyzed through Ultradeep Sequencing

Background Hepatitis B virus reactivation (HBVr) is an important complication of immunosuppressive drug therapy. It can occur via both virological and host factors; however, the underlying mechanisms remain largely unknown. Methods We examined serum samples derived from patients with HBVr and those with acute hepatitis B (AHB). HBV DNA was amplified the targeted nucleic acid molecule and analyzed by next-generation sequencing. Results The percentage of patients infected with genotype Bj among the HBVr patients was significantly higher than that in the AHB patients. The frequency of mutation sites in the whole HBV genome, especially in the envelope region, in the HBVr was significantly higher than that in the AHB. The prevalence of the S3N amino acid substitution in the envelope protein and mutations at positions G1896A and G1899A in the precore region were significantly higher in the HBVr compared to AHB. The population of S3N aa substitution and nt G1896A and G1899A mutations in each individual showed quite a similar percentage of occurrence. Conclusions We identified specific virological factors in patients with HBVr through ultra-deep sequencing. Our findings would be beneficial for the elucidation of mechanisms underlying HBVr development and for disease control.