Full-length Hepatitis B Virus Genome Research Articles

An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable for Genomic Surveillance Within Clinical Diagnostic Settings

Chronic Hepatitis B Virus (HBV) infection remains a significant public health concern, particularly in Africa, where the burden is substantial. HBV is an enveloped virus, classified into ten phylogenetically distinct genotypes (A–J). Tests to determine HBV genotypes are based on full-genome sequencing or reverse hybridization. In practice, both approaches have limitations. Whereas diagnostic sequencing, generally using the Sanger approach, tends to focus only on the S-gene and yields little or no information on intra-patient HBV genetic diversity, reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV diagnostic sequencing protocol suitable for clinical virology that yields both complete genome sequences and extensive intra-patient HBV diversity data. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit (Oxford nanopore Technologies, Oxford, OX4 4DQ, UK), ONT GridION sequencing, genotyping using genome detective software v1.132/1.133, a recombination analysis using jpHMM (26 October 2011 version) and RDP5.61 software, and drug resistance profiling using Geno2pheno v2.0 software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 residual diagnostic samples from HBV-infected patients in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.

The central nervous system is a potential reservoir and possible origin of drug resistance in hepatitis B infection

BackgroundThe significance of hepatitis B virus (HBV) in cerebrospinal fluid (CSF) is unclear. MethodsSynchronous serum and CSF samples were collected from 13 patients. HBV DNA, full-length genome, quasispecies, phylogenetic tree, compartmentalization and mutation of the reverse transcriptase (RT) region were performed based on PCR and sequencing methods. ResultsHBV DNA was detected in the CSF of 3 antiviral-naïve individuals and 1 individual after successful antiviral therapy. Complete full-length HBV genomes were isolated from the CSF of 5 individuals, including 2 with undetectable serum HBV DNA. Ten individuals exhibited distinct CSF-serum quasispecies, 8 harbored independent CSF-serum genetic compartmentalization and phylogenetic trees, and 5 lamivudine/entecavir-associated resistance mutations only in the CSF. The frequencies of rtL180M and rtM204I/V mutations in both serum and CSF were higher in HIV-HBV-coinfected individuals than in the HBV-monoinfected ones (serum: rtL180M: 3.9% vs. 0, P = 0.004; rtM204I/V: 21.3% vs. 0, P < 0.001; CSF: rtL180M: 7.6% vs. 0, P = 0.026; rtM204I/V 7.6% vs. 1.6%, P = 0.097). ConclusionCSF is a potential HBV reservoir, and HBV in CSF harbors distinct evolution and mutation characteristics from those in serum. HIV infection increases the possibility of HBV rtL180M and rtM204I/V mutations in both serum and CSF.

Novel hepatitis B virus reverse transcriptase mutations in patients with sustained viremia despite long-term tenofovir treatment.

Chronic hepatitis B virus (HBV) treatment consists of nucleos(t)ide analogues to suppress viral replication. The HBV inhibitor tenofovir has a high barrier to resistance, however, evidence of virus-escape is emerging. This study investigates HBV evolution in patients undergoing tenofovir treatment with the primary aim to assess the emergence of putative resistance mutations. HBV DNA was extracted from blood samples of two patients with HBeAg-positive chronic HBV infection and persistent viremia despite tenofovir treatment, and subsequently amplified by PCR before full-length HBV genomes were assembled by deep sequencing. The mutation linkage within the viral population was evaluated by clonal analysis of amplicons. Sequence analysis of HBV, derived from 11 samples collected 2010-2020 from one patient, identified 12 non-synonymous single-nucleotide polymorphisms (SNPs) emerging during a tenofovir treatment interruption from 2014 to 2017. Two of the SNPs were in the reverse transcriptase (RT; H35Q and D263E). The two RT mutations were linked and persisted despite restarting tenofovir treatment in 2017. For the second patient, we analyzed HBV derived from six samples collected 2014-2020 following 10 years of tenofovir treatment, and identified five non-synonymous SNPs, that confer resistance towards entecavir and/or lamivudine. Two RT mutations (H35N and P237T) emerged during subsequent 5-year entecavir treatment. H35N was maintained during final tenofovir treatment. Our findings indicate that changes at the conserved residue 35 (H35N/Q) in the HBV RT may be associated with tenofovir resistance. These variants have not previously been described, and further studies are warranted to assess resistance in vitro and in vivo.

OP-2 IN VITRO CHARACTERIZATION OF HEPATITIS B VIRUS REPLICATION AND VIRAL ANTIGEN EXPRESSION AMONG GENOTYPES

Hepatitis B virus (HBV) has been classified into 10 genotypes (A-I) and numerous subgenotypes (SGTs). There is growing evidence that HBV genotypes (GTs) influence clinical outcomes, HBeAg seroconversion rates, severity of liver disease, and response to interferon therapy. However, there is a paucity of data regarding their distinctive biological characteristics, in particular for GTF, the most prevalent in Latin America. To investigate the impact of HBV genotypes on HBV-DNA levels and viral antigen expression. Full-length HBV genomes representing GTs A-D and SGTs F1b and F4 were transfected in Huh7 cell line. Secreted HBeAg and intra and extracellular HBsAg were quantified by EIA. HBV-DNA was analyzed by real-time PCR. Marked differences were observed in HBV replicative capacity as well as HBeAg and HBsAg antigen expression across genotypes (Table 1). GTC secreted significantly higher levels of HBeAg in relation to the other GTs. GTD showed lower HBsAg extracellular levels than all other GTs, while GTA showed the highest HBsAg intracellular levels. Finally, SGTs F1b and F4 showed significantly lower HBV-DNA levels. Regarding the ratio of extra and intracellular HBsAg, GTs A and D showed the lower ratios compared to SGTs F1b and F4, while SGTs F1b and F4 showed the highest HBsAg/HBV-DNA ratio. This study provides new insights into the impact of HBV genotypes on HBV antigen expression and HBV-DNA levels. The uneven expression of antigens, as well as their intracellular accumulation, could be associated with the role of genotypes in pathogenesis. Likewise, the extracellular levels of HBsAg and the replication rate might have implications in immunopathogenesis as well as in the exhaustion of the host's immune system. The virus-cell interaction in different genotypes deserves further study to understand its role in the pathogenesis of HBV infection.

Virome in adult Aedes albopictus captured during different seasons in Guangzhou City, China.

BackgroundThe mosquito Aedes albopictus is an important vector for many pathogens. Understanding the virome in Ae. albopictus is critical for assessing the risk of disease transmission, implementation of vector control measures, and health system strengthening.MethodsIn this study, viral metagenomic and PCR methods were used to reveal the virome in adult Ae. albopictus captured in different areas and during different seasons in Guangzhou, China.ResultsThe viral composition of adult Ae. albopictus varied mainly between seasons. Over 50 viral families were found, which were specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. In rural areas, Siphoviridae (6.5%) was the most common viral family harbored by mosquitoes captured during winter and spring, while Luteoviridae (1.1%) was the most common viral family harbored by mosquitoes captured during summer and autumn. Myoviridae (7.0% and 1.3%) was the most common viral family in mosquitoes captured in urban areas during all seasons. Hepatitis B virus (HBV) was detected by PCR in a female mosquito pool. The first near full-length HBV genome from Ae. albopictus was amplified, which showed a high level of similarity with human HBV genotype B sequences. Human parechovirus (HPeV) was detected in male and female mosquito pools, and the sequences were clustered with HPeV 1 and 3 sequences.ConclusionsLarge numbers of viral species were found in adult Ae. albopictus, including viruses from vertebrates, insects, and plants. The viral composition in Ae. albopictus mainly varied between seasons. Herein, we are the first to report the detection of HPeV and HBV in mosquitoes. This study not only provides valuable information for the control and prevention of mosquito-borne diseases, but it also demonstrates the feasibility of xenosurveillance.Graphical

Comprehensive Analysis of Clinically Significant Hepatitis B Virus Mutations in Relation to Genotype, Subgenotype and Geographic Region

Hepatitis B virus (HBV) is a highly variable DNA virus due to its unique life cycle, which involves an error-prone reverse transcriptase. The high substitution rate drives the evolution of HBV by generating genetic variants upon which selection operates. HBV mutants with clinical implications have been documented worldwide, indicating the potential for spreading and developing their own epidemiology. However, the prevalence of such mutants among the different HBV genotypes and subgenotypes has not been systematically analyzed. In the current study, we performed large-scale analysis of 6,479 full-length HBV genome sequences from genotypes A-H, with the aim of gaining comprehensive insights into the relationships of relevant mutations associated with immune escape, antiviral resistance and hepatocellular carcinoma (HCC) development with HBV (sub)genotypes and geographic regions. Immune escape mutations were detected in 10.7% of the sequences, the most common being I/T126S (1.8%), G145R (1.2%), M133T (1.2%), and Q129R (1.0%). HBV genotype B showed the highest rate of escape mutations (14.7%) while genotype H had no mutations (P < 0.001). HCC-associated mutations were detected in 33.7% of the sequences, with significantly higher frequency of C1653T, T1753V and A1762T/G1764A in genotype G than C (P < 0.001). The overall frequencies of lamivudine-, telbivudine-, adefovir-, and entecavir-resistant mutants were 7.3, 7.2, 0.5, and 0.2%, respectively, while only 0.05% showed reduced susceptibility to tenofovir. In particular, the highest frequency of lamivudine-resistant mutations was observed in genotype G and the lowest frequency in genotype E (32.5 and 0.3%; P < 0.001). The prevalence of HBV mutants was also biased by geographic location, with North America identified as one of the regions with the highest rates of immune escape, antiviral resistance, and HCC-associated mutants. The collective findings were discussed in light of natural selection and the known characteristics of HBV (sub)genotypes. Our data provide relevant information on the prevalence of clinically relevant HBV mutations, which may contribute to further improvement of diagnostic procedures, immunization programs, therapeutic protocols, and disease prognosis.

Epidemiological distribution of hepatitis B virus genotypes in 1–29-year-olds in the mainland of China

BackgroundTo analyze the epidemiological distribution of Hepatitis B virus (HBV) genotype in the mainland of China following the implementation of effective preventive measures. MethodsFive hundred and seventeen HBsAg-positive subjects aged 1–29 years surveyed in the 2014 national HBV sero-survey in the mainland of China were enrolled in the study. The full-length HBV genome was obtained by PCR amplification and sequencing. The HBV genotype was determined by phylogenetic analysis. Combined with questionnaire information, HBV genotype distribution was analyzed. ResultsOf the 517 HBsAg-positive subjects, 369 (71.4%) were included in the analysis. HBV genotypes found were B (45.0%), C (36.6%), D (6.0%), C/D (9.8%), B/C (2.2%), and I (0.5%). Geographic differences in HBV genotype were significant for seven regions. Three serotypes were found: adw (47.2%), adr (35.5%), and ayw (17.3%). B2 (43.9%) and C2 (25.2%) were the two major subgenotypes. The predominant genotypes differed between the Han group and the other ethnic groups. No statistical differences in genotype distribution were found by gender, age group, or hepatitis B (HepB) vaccination history. ConclusionThe prevalence of HBV genotype B was higher than that of genotype C with subgenotypes B2 and C2 endemic in 1–29-year-olds in the mainland of China, after HBV prevalence has reduced significantly due to the implementation of preventive measures. HepB vaccination or other factors did not interfere with HBV genotype distribution. The surveillance of HBV genotype was essential for responding to the potential changes and impact on the preventive policies in the future.

The First Cases of Hepatitis B Virus Subgenotype D4 Detection in Patients with Chronic, Acute, and Occult Hepatitis B in the Russian Federation

To study the molecular genetic structure of hepatitis B virus (HBV) D4 isolates first identified in the Russian Federation. Blood plasma samples of patients from the Republic of Dagestan with previously identified HBV subgenotype D4 were used in the work. For amplification and sequencing reaction, overlapping pairs of specific primers, jointly flanking the entire HBV genome, were used. HBV subgenotype D4 isolates were first detected in the Russian Federation. All 3 isolates were of the ayw2 serotype. Pharmacoresistance mutations L80V and M204I were found in the HIV-infected patient with HBsAg-negative occult HBV. A high frequency of natural polymorphic variants of the Pol gene was shown in all three isolates. In the Precore/Core region, Precore mutations A1846T, C1858T and Core mutations E40D, N74T, N87S, I97F were detected in all 3 isolates. In the X region, all 3 isolates had V5L and P38S mutations. Phylogenetic analysis of the full-length HBV genome showed that the isolates were grouped together with the subgenotype D4 isolate from South Africa. The origin of all three patients with the above isolates from the same geographic region, the phylogenetic proximity of the nucleotide sequences of the complete virus genomes, and a number of identical mutations and polymorphic variants in different genes in all three isolates indicate an independent single import of the virus with the subsequent spread. The routes of distribution, as well as the frequency of HBV D4 occurrence in the Republic of Dagestan, remain unknown and require further research.

Analysis of full-length Hepatitis B Virus Genome From Chronic Hepatitis B-patients With Higher Alanine Aminotransferase Elevation

Background &amp; aim: Higher elevation of alanine aminotransferase (ALT) occasionally leads to severe outcomes in hepatitis B virus (HBV)-infected patients. Our aim is to investigate the HBV sequence mutations associated with higher ALT elevation. Materials &amp; methods: We analyzed full-length HBV sequences from patients with or without higher ALT elevation. Results: Nucleotide mutations in precore and core regions, which are associated with severe hepatitis B, were found in two HBV-infected patients with higher ALT elevation. Amino acid mutations within the pre-S1, pre-S2 and S regions were also found in a patient with HBV virologic breakthrough during the use of nucleoside analogs. Conclusion: It may be useful for HBV-infected patients with higher ALT elevation to analyze full-length HBV genome.

Entecavir-resistant hepatitis B virus decreases surface antigenicity: A full genome and functional characterization.

Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. The rtL180M+rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.

Intra-host diversity of hepatitis B virus during mother-to-child transmission: the X gene may play a key role in virus survival in children after transmission.

Mother-to-child transmission of hepatitis B virus (HBV) is the main route of transmission in Asia, and characterization of HBV quasispecies is needed to further understand virus evolution and adaptation. To understand changes in HBV during mother-to-child transmission, we enrolled nine pairs of mothers and children in the study, including a set of twins. Three groups were infected with HBV genotype C, and six groups were infected with HBV genotype B. The full-length HBV genome was amplified by PCR from serum samples before antiviral treatment, the whole viral genomes from each pair were sequenced, and the complexity and diversity of the quasispecies were analyzed. The entropy of transmitted HBV in children was found to be lower than their mothers, suggesting that there was a bottleneck effect during HBV transmission from the mother to the child. Selective evolution was shown by calculating πN and πS in the whole genomes, and the highest values were obtained for the X gene, which plays a role in viral replication and immune escape. All genotype C patients and only one genotype B pair had a πN/πS greater than 1 ratio, indicating that positive selection had occurred. In addition, quasispecies were found to be different between the twin children despite having the same mother, indicating that virus evolution is host-specific.

The distribution of hepatitis B virus surface antigen polymorphisms at positions associated with vaccine escape.

Hepatitis B virus (HBV) infects over 250 million people worldwide. Vaccination is effective at preventing infection, although several mutations within the "a" determinant region of the HBV surface antigen (HBsAg) are associated with vaccine escape. We evaluated the frequency, genotype, and global distribution of polymorphisms at sites associated with vaccine escape in 4244 unique full-length HBV genomes. The "a" determinant within the Surface gene was inspected for polymorphisms at sites identified previously associated with vaccine escape.Nearly, 268 (6.3%) sequences from 36 countries contained a polymorphism at a site associated with vaccine escape including 22 genotype A, 99 genotype B, 93 genotype C, 32 genotype D, 14 genotype E, 3 genotype F, 2 genotype G, and 3 genotype I. In genotype A, the most common polymorphism occurred at M133. In genotype B, Q129 and M133 occurred 45 and 51 times, respectively, accounting for 94% of polymorphisms. Polymorphisms at G145 were most frequent in genotype C, while P120 was most common in genotype D. Among all genotypes, polymorphisms at M133 were the most common and accounted for 30.9% of polymorphisms. Polymorphisms at T116, P120, F134, K141, and P142 occurred in geographically diverse locations, whereas polymorphisms at Q129, M133, D144, and G145 were concentrated in East Asia. While the sample size is large, this approach relied on convenience sampling within each country, and many countries have no data available, thereby highlighting the need for additional routine surveillance of surface antigen mutations associated with vaccine escape.

Identification of hepatitis B virus genotype A/E recombinants in Ghana.

Hepatitis B virus (HBV) exhibits a high degree of heterogeneity with at least 10 genotypes (A-J) identified to date. Intergenotypic recombination is relatively common. Previously, we investigated HBV drug resistance in HIV/HBV co-infected individuals in Ghana. After identifying multiple circulating genotypes and a novel D/E recombinant, we sought to determine if additional individuals were also infected with recombinant HBV. Partial genome sequences from three individuals were initially identified as genotype A4. Full-length HBV genomes were obtained using rolling circle amplification followed by PCR and shown to cluster with known A/E recombinant viruses. Similar recombination breakpoints were observed in these three individuals suggesting local spread of this novel recombinant HBV in Ghana.

Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line

Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro.

Clinical and virological implications of A1846T and C1913A/G mutations of hepatitis B virus genome in severe liver diseases

Objective: Mutations occurring within different genes of hepatitis B virus (HBV) genome may have different clinical implications. This study aimed to observe the clinical and virological implications of the A1846T and C1913A/G mutations of HBV genome in the development and treatment outcome of severe liver diseases, which has not been previously determined.Materials and methods: A total of 438 cases of patients with liver diseases were retrospectively reviewed, including 146 with mild chronic hepatitis B infection (CHB-M), 146 with severe chronic hepatitis B infection (CHB-S), and 146 with acute-on-chronic liver failure (ACLF). Partial or full-length HBV genome was directly sequenced. Replicons containing A1846T, C1913A or other mutant sequences, or the wild-type counterparts were constructed respectively, and then transfected into HepG2 cells for phenotype analysis.Results: There was significant difference in the detection rates of A1846T (30.82%, 40.41% and 55.48%, respectively) and C1913A/G (15.52%, 28.77%, and 35.62%, respectively) among patients with CHB-M, those with CHB-S, and those with ACLF (p < .01). A1846T was significantly associated with the mortality of ACLF patients within six months after the disease onset (OR 1.704, p = .041). In vitro experiment revealed that A1846T mutant resulted in 3.20-fold and 1.85-fold increase of replication capacity and promoter activity, respectively compared with wild type counterpart (p < .001), while C1913A led to a significant decrease of core protein expression (p < .05).Conclusion: Occurrence of A1846T and C1913A is positively associated with clinical presentations of severe liver disease. A1846T mutation is significantly associated with poor prognosis of ACLF.

Transcription and regulation of hepatitis B virus genes in host sperm cells

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

Profile of Mutations in the Reverse Transcriptase and Overlapping Surface Genes of Hepatitis B Virus (HBV) in Treatment-Naïve Indonesian HBV Carriers

Mutations in the reverse transcriptase (RT) region of the hepatitis B virus (HBV) genome are an important factor in low therapeutic effectiveness. Nonetheless, the prevalence of these mutations in HBV strains isolated previously in Indonesia has not been systematically examined. Therefore, in this study, we investigated the profile of mutations in the RT region and the associations of these mutations with amino acid changes in the surface protein in the virus of treatment-naïve Indonesian HBV carriers. Overall, 96 sequences of the full-length Indonesian HBV genomes (genotype B, n = 54; genotype C, n = 42) were retrieved from the National Center for Biotechnology Information. Naturally occurring primary and/or compensatory drug resistance mutations were found in 6/54 (11.1%) genotype B strains and in 1/42 (2.4%) genotype C strains. The potential mutations underlying resistance to a nucleos(t)ide analog and/or pretreatment mutations were more frequent in both genotypes but more frequent in genotype C strains than in genotype B strains. The A-B interdomain region in the RT gene was more frequently mutated in genotype C than in genotype B (3.51 ± 2.53 vs. 1.08 ± 1.52, P ＜ 0.001). Knowledge about the mutational profiles of the RT gene and changes in the surface protein may help clinicians to select the most appropriate antiviral drug and vaccination or HBV immunoglobulin regimen for management of HBV infection in Indonesia.

Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization.

Hepatitis B virus (HBV) infection can be associated with a spectrum of clinical outcomes. Transient transfection of the clinical HBV isolates in human hepatoma cell lines can establish their biological properties to shed light on their different pathogenic potentials, yet very few clinical HBV isolates have been functionally characterized so far. The technical challenges include faithful amplification of the full-length HBV genome from clinical samples and conversion into a replication-competent form. We have improved a published method to amplify the full-length HBV genome from blood samples. Two alternative approaches are used to render the cloned HBV genome replication competent: release and circularization of the 3.2-kb HBV genome prior to each transfection experiment or conversion of the monomeric clone into a tandem dimer version.

Characteristics of CpG Islands and their quasispecies of full-length hepatitis B virus genomes from patients at different phases of infection

BackgroundCpG islands in hepatitis B virus (HBV) genome are potential targets for methylation mediated gene silencing, and may be involved in the pathogenesis of HBV infection. To date, their characteristics in HBV quasispecies (QS) remain largely unknown. The purpose of this study was to investigate the characteristics of CpG islands in HBV QS. MethodsForty patients diagnosed as acute hepatitis B (AHB, n = 10), immune-tolerant HBV carriers (IT, n = 9), chronic hepatitis B (CHB, n = 11), or acute on chronic liver failure (ACLF, n = 10), were enrolled in this case–control study. A total of 599 clones were isolated, and full-length HBV genomes were sequenced.ResultsCpG island II (CGII) in AHB group was shorter in length and its QS heterogeneity was lower than that in the chronic infection group. Among the chronic infection subgroups, CGII and CpG island III (CGIII) in IT group were longer and their heterogeneity was lower compared to CHB and ACLF groups. Length of CGII correlated with HBV DNA levels positively while the complexity and diversity of CGII correlated with HBV DNA levels negatively. Moreover, CGII and CGIII were shorter in genotype B than those in genotype C, while QS complexity and diversity of either CGII or CGIII had no significant difference between genotype B and C.ConclusionsOverall, our results suggest that the distribution, length and QS heterogeneity of CpG islands in full-length HBV genome differ across clinical phases of infection, of which the mechanism warrants further study.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3192-3) contains supplementary material, which is available to authorized users.

Human immunodeficiency virus and hepatitis B genotype G/A2 recombinant co-infection: a case study.

BackgroundHepatitis B virus (HBV) genotypes have distinct geographical distributions and are associated with different clinical courses. HBV genotype G (HBV/G) is extremely rare among Human immunodeficiency virus (HIV) infected populations in Japan. Genetic analysis and clinical course of recombinant forms with HBV/G infection are seldom reported in the literature.Case presentationA 36-year old homosexual man with HIV infection was referred to a general hospital for assessment of chronic HBV infection. We cloned full-length HBV isolates and determined the complete genome sequences of 2 obtained clones, although mixture of multiple variant with different length is detected by HBV-DNA genotyping. The Bootscaning analysis using a full-length HBV genome revealed the clones represented as the HBV/A2 and the HBV/G/A2 recombinant strain. The HBV-DNA decreased from >9.1 to 2.5 log copies/mL after 24 months of antiretroviral therapy.ConclusionsThis patient was co-infected with HBV/A2 and HBV/G/A2 recombinant strain. This recombinant strain was not identical to HBV/G/A2 strains previously reported from Japan. Recombination with other genotypes could alter the clinical manifestations of chronic hepatitis B in people living with HIV.