Search

Summary Background & Aims We aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS). Methods Fifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies. Results HBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p 1 year after seroconversion (0.0103 vs. 0.0068, p, Graphical abstract, Highlights • Deep sequencing of whole HBV genome uncovers the quasispecies changes in chronic hepatitis B patients. • The nucleotide diversity of HBV negatively correlates with viraemia during HBeAg loss/seroconversion. • Viral quasispecies diversity is greater in spontaneous HBeAg seroconverters before and after seroconversion than in treatment-naïve non-seroconverters. • Responders to IFN have greater viral quasispecies diversity than non-responders at 24 weeks after treatment. • The genome positions of non-synonymous intra-host single nucleotide variants (iSNVs) of HBV tend to be located at possible T cell epitopes.

Exogenous 3-hydroxybutyrate can be utilized by a variety of soil bacteria as a carbon and energy source. However, the membrane transporter responsible for 3-hydroxybutyrate uptake remains unidentified. The Bacillus subtilis strain 168 gene yxjC (herein renamed hbuT) encodes a putative gluconate transporter GntT-type membrane transporter with a previously unknown function. hbuT is organized within the same operon with genes that are used for metabolism of 3-hydroxybutyrate. Here we report that a null mutation of hbuT reduced uptake of 3-hydroxybutyrate by B. subtilis cells grown in nutrient sporulation medium. The SigE-controlled HbuT transporter apparently plays a major role in the uptake of 3-hydroxybutyrate. Uptake of 3-hydroxybutyrate by the HbuT transporter occurred in a specific manner at the early sporulation stage. SigE-controlled hbuT expression and 3-hydroxybutyrate uptake were also subject to CcpA-mediated glucose repression. hbuT expression was not induced by exogenous 3-hydroxybutyrate and B. subtilis cells could not utilize 3-hydroxybutyrate as a sole carbon source for growth. HbuT homologs are present in a wide variety of Gram-positive Bacillus species, some Gram-negative Acinetobacter species and a small group of other bacteria. This is the first tentative identification of a membrane transporter responsible for the uptake of 3-hydroxybutyrate in bacteria.

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

Precore (PC) (G1896A) and basal core promoter (BCP) (A1762T/G1764A) mutations of the hepatitis B virus (HBV) genome often emerge in chronic hepatitis B (CHB) patients. Their roles in hepatitis B e antigen (HBeAg) seroconversion induced by interferon (IFN) therapy remain controversial, partly because quantitative analysis for these mutants is lacking. This study aimed to develop a new assay to accurately quantify the PC and BCP mutant percentages and correlate their dynamic changes with IFN-induced HBeAg seroconversion in HBeAg-positive CHB patients. The PC and BCP mutant percentages were analyzed by polymerase chain reaction (PCR)-pyrosequencing. Our results showed that this quantitative assay for PC and BCP mutants achieved high accuracy (R2 > 0.99) within a range between 10% and 90% mutants. We examined dynamic changes of the PC and BCP mutant percentages following IFN treatment in 203 HBeAg-positive CHB patients. By multiple logistic regression analysis, we found that the chance of HBeAg seroconversion increased by 2.2% (odds ratio [OR] = 1.022, 95% confidence interval [CI]: 1.009-1.034, P = 0.001) and 2.3% (OR = 1.023, 95% CI: 1.010-1.037, P = 0.001) per 1% increase of the pretreatment PC and BCP mutant percentages, respectively, after adjustment for other predictors. However, only the pretreatment PC mutation percentage was significantly associated with HBeAg seroconversion with HBV DNA < 2,000 IU/mL (OR = 1.030, 95% CI: 1.014-1.047, P < 0.001). Furthermore, the mutant percentage of PC, but not BCP, in patients achieving HBeAg seroclearance with HBV DNA < 20,000 IU/mL increased significantly during IFN treatment (P = 0.039). Interestingly, patients with HBeAg seroconversion who had a high PC mutant percentage at the end of IFN treatment tended to exhibit high viremia after seroconversion. Conclusion: Quantitative analysis of PC and BCP mutants can predict IFN-induced HBeAg seroconversion and demonstrate their distinct evolution patterns during HBeAg seroconversion. (HEPATOLOGY 2013)