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.

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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 <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome. Conclusions Higher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome. Lay summary HBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

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.