Hurng-Yi Wang

The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo

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.

Adaptive genic evolution in the Drosophila genomes

Determining the extent of adaptive evolution at the genomic level is central to our understanding of molecular evolution. A suitable observation for this purpose would consist of polymorphic data on a large and unbiased collection of genes from two closely related species, each having a large and stable population. In this study, we sequenced 419 genes from 24 lines of Drosophila melanogaster and its close relatives. Together with data from Drosophila simulans, these data reveal the following. (i) Approximately 10% of the loci in regions of normal recombination are much less polymorphic at silent sites than expected, hinting at the action of selective sweeps. (ii) The level of polymorphism is negatively correlated with the rate of nonsynonymous divergence across loci. Thus, even under strict neutrality, the ratio of amino acid to silent nucleotide changes (A:S) between Drosophila species is expected to be 25–40% higher than the A:S ratio for polymorphism when data are pooled across the genome. (iii) The observed A/S ratio between species among the 419 loci is 28.9% higher than the (adjusted) neutral expectation. We estimate that nearly 30% of the amino acid substitutions between D. melanogaster and its close relatives were adaptive. (iv) This signature of adaptive evolution is observable only in regions of normal recombination. Hence, the low level of polymorphism observed in regions of reduced recombination may not be driven primarily by positive selection. Finally, we discuss the theories and data pertaining to the interpretation of adaptive evolution in genomic studies.

Molecular and Phylogenetic Analyses Suggest an Additional Hepatitis B Virus Genotype “I”

A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype.

Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data

We present the analysis of the evolution of tumors in a case of hepatocellular carcinoma. This case is particularly informative about cancer growth dynamics and the underlying driving mutations. We sampled nine different sections from three tumors and seven more sections from the adjacent nontumor tissues. Selected sections were subjected to exon as well as whole-genome sequencing. Putative somatic mutations were then individually validated across all 9 tumor and 7 nontumor sections. Among the mutations validated, 24 were amino acid changes; in addition, 22 large indels/copy number variants (>1 Mb) were detected. These somatic mutations define four evolutionary lineages among tumor cells. Separate evolution and expansion of these lineages were recent and rapid, each apparently having only one lineage-specific protein-coding mutation. Hence, by using a cell-population genetic definition, this approach identified three coding changes (CCNG1, P62, and an indel/fusion gene) as tumor driver mutations. These three mutations, affecting cell cycle control and apoptosis, are functionally distinct from mutations that accumulated earlier, many of which are involved in inflammation/immunity or cell anchoring. These distinct functions of mutations at different stages may reflect the genetic interactions underlying tumor growth.

Identification of androgen response elements in the enhancer I of hepatitis B virus: A mechanism for sex disparity in chronic hepatitis B

Hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) occurs more often in men than in women. Male HBV carriers usually have higher viral loads, which is a well‐known risk factor for HCC. Whether and how the male androgen axis regulates HBV transcription and replication is investigated here. We used HBV transgenic mice to evaluate any sex disparity of serum hepatitis B surface antigen and HBV titers as well as the castration effect on this disparity. Compared to females, HBV transgenic male mice showed higher hepatitis B surface antigen and viral titers, which were lessened by castration of the males. In a cell culture system, HepG2 cells transfected with HBV and androgen receptor (AR) constructs were used to study the effect of the androgen pathway on viral transcription and replication. We found the ligand‐stimulated AR could increase the transcription of HBV RNAs through its transcription activation domain. A genomic region within HBV enhancer I was identified that is responsible for the transcriptional activation of AR. The results from chromatin immunoprecipitation and in vitro binding assays further demonstrated a direct binding of AR to this region, in a ligand‐dependent manner. Two androgen‐responsive element motifs in this region were identified, and their mutations can significantly abolish the AR effects. Conclusion: This study demonstrated that the androgen pathway can increase the transcription of HBV through direct binding to the androgen‐responsive element sites in viral enhancer I. This may explain a higher HBV titer in male carriers and an increased risk of HCC. (HEPATOLOGY 2009.)

Rate of Evolution in Brain-Expressed Genes in Humans and Other Primates

Brain-expressed genes are known to evolve slowly in mammals. Nevertheless, since brains of higher primates have evolved rapidly, one might expect acceleration in DNA sequence evolution in their brain-expressed genes. In this study, we carried out full-length cDNA sequencing on the brain transcriptome of an Old World monkey (OWM) and then conducted three-way comparisons among (i) mouse, OWM, and human, and (ii) OWM, chimpanzee, and human. Although brain-expressed genes indeed appear to evolve more rapidly in species with more advanced brains (apes > OWM > mouse), a similar lineage effect is observable for most other genes. The broad inclusion of genes in the reference set to represent the genomic average is therefore critical to this type of analysis. Calibrated against the genomic average, the rate of evolution among brain-expressed genes is probably lower (or at most equal) in humans than in chimpanzee and OWM. Interestingly, the trend of slow evolution in coding sequence is no less pronounced among brain-specific genes, vis-à-vis brain-expressed genes in general. The human brain may thus differ from those of our close relatives in two opposite directions: (i) faster evolution in gene expression, and (ii) a likely slowdown in the evolution of protein sequences. Possible explanations and hypotheses are discussed.

Distinct Hepatitis B Virus Dynamics in the Immunotolerant and Early Immunoclearance Phases

ABSTRACT Little is known about hepatitis B virus (HBV) diversity changes within a host during the immunotolerant phase of chronic HBV infection. Such knowledge, nevertheless, may help in understanding how host immunity and HBV interact at the early stage of infection. In this study, serial serum samples were collected from a long-term (>17 years) follow-up cohort of seven patients, and multiple copies of the full-length viral genome from serially sampled sera were recovered and analyzed. Viral genetic diversity was positively correlated with host immunity, represented by levels of alanine aminotransferase (ALT), but was negatively correlated with the viral copy number. During the immunotolerant phase, when the host immunity was feeble (ALT < 20 U/liter), viral nucleotide diversity decreased while copy numbers increased. Rates of evolutionary change derived for different patients were in a very narrow range (1.6 × 10−5 to 5.4 × 10−5/site/year). As the disease progressed toward the immunoclearance phase (ALT > 20 U/liter), viral diversity increased but copy numbers decreased. Evolutionary rates varied among patients in accordance with their levels of ALT, ranging from 9.6 × 10−6 to 3.2 × 10−4/site/year. More than half (19/32 sites) of positively selected sites resided in immune epitopes, suggesting their possible role in host immunity. Our results demonstrate that host immunity is a dominant factor in HBV evolution. Different selective forces, including immune-mediated positive selection and virus-mediated negative selection, operate in tandem in shaping viral population dynamics within a host.

Influence of glaciation on divergence patterns of the endemic minnow, Zacco pachycephalus, in Taiwan

The genetic subdivision of the endemic minnow, Zacco pachycephalus, in 12 rivers of Taiwan was studied by allozyme electrophoresis. Among 26 loci surveyed, six were significantly differentiated among sites. Allozyme genotype frequencies within samples accord with Hardy–Weinberg expectations. A highly divergent genetic structure among sites (FST = 0.497) with low genetic variability within samples (H = 0.006–0.062, mean = 0.03) suggests that local populations originated from a small number of founders. upgma and Fitch trees derived from Nei’s genetic distance and hierarchical analysis of FST values reveal that samples of Z. pachycephalus can be divided into northern, middle, and southern subgroups and that most genetic variation (87%) is distributed among rather than within groups. It is proposed that there were two isolated refugia during the most recent glacial period, and that populations of the southern group (Group I) originated from a southern refugium in Taiwan below the boundary of the Kaoping River, and other groups (Group II), including northern and middle subgroups, originated from a refugium located on the extinct landbridge between Taiwan and mainland China. The postglacial division between northern and middle subgroups may be due to hydrological differences between these two regions.

Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota

Significance Ninety-five percent of adult-acquired infections lead to spontaneous clearance, whereas >90% of exposed neonates and ∼30% of children aged 1–5 y fail to resolve hepatitis B virus (HBV) and develop chronic infection. A strong, diverse, adaptive immune response is considered essential for HBV clearance, but the mechanisms to generate a favorable response remain elusive. Here, we show that, while 12-wk-old C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their 6-wk-old counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented mice from rapidly clearing HBV. This model is valuable to the study of liver tolerance and enables the investigation of the mechanisms involved in effective control of HBV. A unique feature of hepatitis B virus (HBV) infection in humans is that viral clearance heavily depends on the age of exposure. However, the reason for this remains unclear. Here we show that gut microbiota contribute to the age dependence of HBV immunity in a hydrodynamic transfection mouse model. Although adult (12-wk-old) C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their young (6-wk-old) counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented adult mice from rapidly clearing HBV. Young mice with the Toll-like-receptor (TLR) 4 mutation (C3H/HeJ) exhibited rapid HBV clearance. The results suggest that an immuno-tolerating pathway to HBV prevailed in young mice, before the establishment of gut bacteria, through a TLR4-dependent pathway and that the maturation of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance.

Phylogeny, taxonomy, and biogeography of the oriental pitvipers of the genus Trimeresurus (Reptilia: Viperidae: Crotalinae): a molecular perspective.

Abstract Based on sequence variation in 806 bp of the mitochondrial 12S rRNA gene, phylogenetic relationships were inferred for 14 species of Trimeresurus (sensu lato) including all East Asian members. Samples analyzed also included representatives of all assemblages of species that are frequently treated as separate genera except for T. mangshanensis, a type species of the recently described monotypic genus Ermia. Results support some previous accounts chiefly from morphological studies, such as distinct divergence of T. wagleri from the remainder, and monophyly of T. mucrosquamatus, T. flavoviridis, T. jerdonii, T. elegans and T. tokarensis. On the other hand, our results negated a putative close affinity of T. monticola and T. okinavensis, and indicated the sister relationship of the latter with T. gracilis. Phylogenetic relationships revealed in this study suggested that the genus Trimeresurus dispersed into the Ryukyu region at least three times, and that T. flavoviridis and T. tokarensis from the central Ryukyus constitute a relict clade.