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...

Transcriptome analysis of hepatoma cells transfected with Basal Core Promoter (BCP) and Pre-Core (PC) mutant hepatitis B virus full genome construct

Infections with Basal Core Promoter (BCP) (A1762T/G1764A) and Pre-Core (PC) (G1896A) hepatitis B virus HBeAg mutants are associated with severe liver injury. We analysed host cell responses in HepG2/C3A, hepatoma cells transfected with infectious clones developed from genotype D wild type (WT) and BCP/PC mutant (MT) viruses isolated from an acute resolved and an acute liver failure hepatitis B case respectively. Cells transfected with MT virus construct showed ~55 % apoptosis and with WT ~30 % apoptosis at 72 h. To determine possible roles of HBe and HBx proteins in apoptosis, we cloned these genes and co-transfected cells with WT+HBe/HBx or MT+HBe/HBx constructs. Co-expression of HBe protein improved cell viability significantly in both WT and MT virus constructs, indicating an important role of HBe in protecting cells. RNA sequencing analysis carried out at 12 and 72 h post-transfection with WT virus construct showed enrichment of innate/adaptive immune response-activating signal transduction, cell survival and amino acid/nucleic acid biosynthetic pathways at 12 and 72 h. By contrast, MT virus construct showed enrichment in host defence pathways and some biosynthetic pathways at the early time point (12 h), and inflammatory response, secretary granule, regulation of membrane potential and stress response regulatory pathways at the late time point (72 h). There was a significant down-regulation of genes involved in endoplasmic reticulum and mitochondrial functions and metabolism with MT construct and this possibly led to induction of apoptosis in cells. Considering rapid apoptotic changes in cells transfected with MT construct, it can be speculated that HBeAg plays a crucial role in cell survival. It enhances induction of metabolic and synthetic pathways and facilitates management of cellular stress that is induced due to hepatitis B virus infection/replication.

Global Occurrence of Clinically Relevant Hepatitis B Virus Variants as Found by Analysis of Publicly Available Sequencing Data

Several viral factors impact the natural course of hepatitis B virus (HBV) infection, the sensitivity of diagnostic tests, or treatment response to interferon-α and nucleos(t)ide analogues. These factors include the viral genotype and serotype but also mutations affecting the HBV surface antigen, basal core promoter/pre-core region, or reverse transcriptase. However, a comprehensive overview of the distribution of HBV variants between HBV genotypes or different geographical locations is lacking. To address this, we performed an in silico analysis of publicly available HBV full-length genome sequences. We found that not only the serotype frequency but also the majority of clinically relevant mutations are primarily associated with specific genotypes. Distinct mutations enriched in certain world regions are not explained by the local genotype distribution. Two HBV variants previously identified to confer resistance to the nucleotide analogue tenofovir in vitro were not identified, questioning their translational relevance. In summary, our work elucidates the differences in the clinical manifestation of HBV infection observed between genotypes and geographical locations and furthermore helps identify suitable diagnostic tests and therapies.

The effect of basal core promoter and pre-core mutations on HBV replication and persistence in mice

The appearance of the BCP or Pre-C mutations, which reduce or abolish HBeAg production, could increase HBV replication. The remove of the HBeAg often lead to a vigorous immune response, which has an important role in HBV related fulminant outcome. In this study, BCP mutations and Pre-C mutations were separately introduced by site-directed mutagenesis in the same genetic background of an HBV infectious clone, to determine the effect of these mutations per se on replication. BCP and Pre-C mutations increased HBV replication both in vitro and in vivo. HBV could persist in mice injected with wild type HBV infectious clone for about 7 weeks. However, HBV could persist about 5 weeks in mice injected with BCP HBV infectious clone, and 3 weeks only in mice injected with Pre-C HBV infectious clone. HBV related CD8+ CTL response in BCP HBV infectious clone injected mice only slightly increased, but significantly increased in Pre-C HBV infectious clone injected mice compared with that in wild type HBV infectious clone injected mice. The population of Tregs significantly increased in liver but not in spleen of mice injected with Pre-C HBV infectious clone. In summary, we demonstrate that HBeAg plays an important role in suppressing the CTL response, which is related with increasing the frequency of Tregs in mouse. Lack of HBeAg expression leads to the partial loss of immune tolerance.

Impacts of the Percentage of Basal Core Promoter Mutation on the Progression of Liver Fibrosis After Hepatitis B e Antigen Seroconversion

Background We investigated the relationships among the percentage of hepatitis B virus (HBV) mutations and liver fibrosis after hepatitis B e antigen (HBeAg) seroconversion. Methods We quantified the percentage of HBV mutants by pyrosequencing using serum samples obtained at inflammatory phase and after HBeAg seroconversion in 160 initially HBeAg-positive chronic HBV-infected patients. The relationships between antiviral agents, percentages of HBV mutations, and liver stiffness measurements (LSMs) were analyzed. Results We demonstrated that the percentages of A1762T/G1764A mutation are significantly higher in subjects with an LSM >7 kPa than in those with an LSM ≤7 kPa after HBeAg seroconversion. Hepatitis B e antigen seroconversion age is positively correlated with the percentages of A1762T/G1764A mutation at inflammatory phase before HBeAg seroconversion. Subjects who underwent interferon, entecavir, or tenofovir disoproxil fumarate therapy before HBeAg seroconversion possessed a lower percentage of A1762T/G1764A mutation after HBeAg seroconversion. The percentage of A1762T/G1764A ≥20% after HBeAg seroconversion was predictive of an LSM >7 kPa (hazard ratio = 6.37, P = .001). The presence of A1762T/G1764A led to downregulated messenger ribonucleic acid and protein levels of programmed-death ligand-1 (PD-L1) in hepatocytes. Conclusions The percentage of A1762T/G1764A mutations after HBeAg seroconversion was associated with liver fibrosis. The A1762T/G1764A mutation may evoke hepatic inflammation by suppressing PD-L1 in hepatocytes.

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.