Full-length Hepatitis B Virus 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.

Synthesis of the full-length hepatitis B virus core protein and its capsid formation.

Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.

Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population.

Hepatitis B virus (HBV) genotype C is a prevalent HBV genotype in the Chinese population. Although genotype C shows higher sequence heterogeneity and more severe liver disease than other genotypes, its pathogenesis and immunological traits are not yet fully elucidated. In this study, we first established and chemically synthesized the consensus sequence based on representative 138 full-length HBV genotype C genomes from the Chinese population. The pHBV1.3C plasmid system, containing a 1.3-fold full-length HBV genotype C consensus sequence, was constructed for subsequent validation. Next, we performed functional assays to investigate the replicative competence of pHBV1.3C in vitro through the transient transfection of HepG2 and Huh7 cells and validated the in vivo function via a hydrodynamic injection to BALB/c recipient mice. The in vitro investigation revealed that the extracellular HBV DNA and intracellular replicative intermediate (i.e., pregenomic RNA, pgRNA) were apparently measurable at 48 h, and the HBsAg and HBcAg were still positive in hepatoma cells at 96 h. We also found that HBsAg and HBeAg accumulated at the extracellular and intracellular levels in a time-dependent manner. The in vivo validation demonstrated that pHBV1.3C plasmids induced HBV viremia, triggered morphological changes and HBsAg- or HBcAg- positivity of hepatocytes, and ultimately caused inflammatory infiltration and focal or piecemeal necrosis in the livers of the murine recipients. HBV protein (HBsAg) colocalized with CD8+ T cells or CD4+ T cells in the liver. F4/80+ Kupffer cells were abundantly recruited around the altered murine hepatocytes. Taken together, our results indicate that the synthetic consensus sequence of HBV genotype C is replication-competent in vitro and in vivo. This genotype C consensus genome supports the full HBV life cycle, which is conducive to studying its pathogenesis and immune response, screening novel antiviral agents, and further optimizing testing and therapeutics.

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.

Hepatitis B Virus Genotype E/A Recombinants from Blood Donors in Beira, Mozambique.

Hepatitis B virus (HBV) is endemic in many parts of sub-Saharan Africa. Here, we present 5 full-length HBV recombinant genomes from blood donors in Beira, Mozambique. The genomes are recombinants between genotypes E and A and are distantly related to one another, based on their genetic distances.

Hepatitis B virus suppresses complement C9 synthesis by limiting the availability of transcription factor USF-1 and inhibits formation of membrane attack complex: implications in disease pathogenesis

BackgroundThe complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis.MethodsBlood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed.ResultsAmong the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients.ConclusionCollectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.

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.

Distribution of hepatitis B virus genotypes and subgenotypes: A meta-analysis.

Hepatitis B virus (HBV) genotypes and subgenotypes have distinct geographical distributions and influence a number of clinical disease features and responses to treatment. There are many reports on the distribution of HBV genotypes, but great differences are present between studies. What's more, a meta-analysis of HBV genotype- and subgenotype-distribution by country is lacking.A comprehensive literature search was performed in PubMed and a systematic search of full-length HBV sequences and S gene sequences was conducted in the GenBank database. HBV genotypes were checked and subgenotypes were determined by phylogenetic comparison of full-length HBV sequences or S gene sequences. STATA 12.0 was used for the analysis for countries with multiple datasets. BEAST 2.5.2 was used for Bayesian phylogenetic analysis to infer the evolutionary time scales of HBV.This study includes 309 datasets from 110 countries, including 188 relevant studies, 58 full-length gene datasets, and 63 S gene datasets. The meta-analysis was performed on 274 datasets from 75 countries. The distribution of genotypes is more detailed than those described by previous studies. While the overall genotype distribution is similar to that reported in previous studies, some notable aspects were different. The main genotypes present in south-eastern Africa, North Africa, and West Africa are genotypes A, D, and E, respectively. Genotypes G and H are mainly distributed in Mexico. Genotype F is mainly distributed in central and South America, but genotypes A and D are also common in Brazil, Cuba, and Haiti.This study provides a more accurate description of the distribution of HBV genotypes and subgenotypes in different countries and suggests that the differences in genotype distribution may be related to ethnicity and human migration.

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

Origins and Evolution of the Primate Hepatitis B Virus.

Recent interest in the origins and subsequent evolution of the hepatitis B virus (HBV) has strengthened with the discovery of ancient HBV sequences in fossilized remains of humans dating back to the Neolithic period around 7,000 years ago. Metagenomic analysis identified a number of African non-human primate HBV sequences in the oldest samples collected, indicating that human HBV may have at some stage, evolved in Africa following zoonotic transmissions from higher primates. Ancestral genotype A and D isolates were also discovered from the Bronze Age, not in Africa but rather Eurasia, implying a more complex evolutionary and migratory history for HBV than previously recognized. Most full-length ancient HBV sequences exhibited features of inter genotypic recombination, confirming the importance of recombination and the mutation rate of the error-prone viral replicase as drivers for successful HBV evolution. A model for the origin and evolution of HBV is proposed, which includes multiple cross-species transmissions and favors subsequent recombination events that result in a pathogen and can successfully transmit and cause persistent infection in the primate host.

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.

Characterization of mutations in the reverse transcriptase region of hepatitis B virus in treated and untreated chronic hepatitis B patients.

The reverse transcriptase (RT) region of the hepatitis B virus (HBV) is the target of antiviral treatment. However, the discrepancy in RT mutations between nucleos(t)ide analogue (NA)-treated and -untreated chronic hepatitis B (CHB) patients is un clear. Serum samples were collected from 119 NA-treated and 135 NA-untreated patients. The sampling time was decided by the clinician. Full-length HBV RT regions were amplified using nest polymerase chain reaction. The mutations within the RT region were analysed by direct sequencing. The incidence of RT mutations in treated patients was higher than that in untreated patients (p<0.05). The classic drug-resistant mutations were detected in 44.5% (53/119) of treated patients, which was significantly higher than in untreated patients (6.7% [9/135]) (p<0.05). The non-classical mutations showed their complexity and diversity in both patient groups. Multiple mutations (three or more) were more frequent in treated patients than in untreated patients (p<0.05). Several novel mutations might be related to NA resistance. The selection pressures of NAs accelerated the development of RT mutations, especially within the functional domain. Mutations in the RT region occurred not only at classical sites, but also at other non-classical sites, which might be related to drug resistance and/or viral replication. The biological function and fitness of HBV isolates harbouring these novel mutations need further in vitro and in vivo verification experiments.

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.

Assembly of Capsids from Hepatitis B Virus Core Protein Progresses through Highly Populated Intermediates in the Presence and Absence of RNA.

The genetic material of viruses is protected by protein shells that are assembled from a large number of subunits in a process that is efficient and robust. Many of the mechanistic details underpinning efficient assembly of virus capsids are still unknown. The assembly mechanism of hepatitis B capsids has been intensively researched using a truncated core protein lacking the C-terminal domain responsible for binding genomic RNA. To resolve the assembly intermediates of hepatitis B virus (HBV), we studied the formation of nucleocapsids and empty capsids from full-length hepatitis B core proteins, using time-resolved small-angle X-ray scattering. We developed a detailed structural model of the HBV capsid assembly process using a combination of analysis with multivariate curve resolution, structural modeling, and Bayesian ensemble inference. The detailed structural analysis supports an assembly pathway that proceeds through the formation of two highly populated intermediates, a trimer of dimers and a partially closed shell consisting of around 40 dimers. These intermediates are on-path, transient and efficiently convert into fully formed capsids. In the presence of an RNA oligo that binds specifically to the C-terminal domain the assembly proceeds via a similar mechanism to that in the absence of nucleic acids. Comparisons between truncated and full-length HBV capsid proteins reveal that the unstructured C-terminal domain has a significant impact on the assembly process and is required to obtain a more complete mechanistic understanding of HBV capsid formation. These results also illustrate how combining scattering information from different time-points during time-resolved experiments can be utilized to derive a structural model of protein self-assembly pathways.

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.