Recent advances in the study of hepatitis B virus covalently closed circular DNA

Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.

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Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

A nucleic acid biosensor for the detection of a short sequence related to the hepatitis B virus using bis(benzimidazole)cadmium(II) dinitrate as an electrochemical indicator

A novel hybridization indicator, bis(benzimidazole)cadmium(II) dinitrate (Cd(bzim)(2)(NO(3))(2)), was utilized to develop an electrochemical DNA biosensor for the detection of a short DNA sequence related to the hepatitis B virus (HBV). The sensor relies on the immobilization and hybridization of the 21-mer single-stranded oligonucleotide from the HBV long repeat at the glassy carbon electrode (GCE). The hybridization between the probe and its complementary sequence as the target was studied by enhancement of the peak of the Cd(bzim)(2)(2+) indicator using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay time. With this approach, a sequence of the HBV could be quantified over the range from 1.49x10(-7)M to 1.06x10(-6)M, with a linear correlation of r=0.9973 and a detection limit of 8.4x10(-8)M. The Cd(bzim)(2)(2+) signal observed from the probe sequence before and after hybridization with a four-base mismatch containing sequence was lower than that observed after hybridization with a complementary sequence, showing good selectivity. These results demonstrate that the Cd(bzim)(2)(2+) indicator provides great promise for the rapid and specific measurement of the target DNA.