Precore Mutations Research Articles

Impaired HBsAg release and antiproliferative/antioxidant cell regulation by HBeAg-negative patient isolates reflects an evolutionary process.

The hepatitis B e antigen (HBeAg)-negative infection Phase 3 is characterized by no or minimal signs of hepatic inflammation and the absence of hepatic fibrosis. However, underlying molecular mechanisms leading to this benign phenotype are poorly understood. Genotype A, B and D HBeAg-negative patient isolates with precore mutation G1896A from Phase 3 were analysed in comparison with respective HBeAg-positive rescue mutant and HBeAg-positive wild-type reference genomes regarding differences in viral replication, morphogenesis, infectivity and impact on NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-dependent gene expression and cellular kinome. In comparison with reference genomes, the patient isolates are characterized by a lower intra- and extracellular hepatitis B surface antigen (HBsAg)-amount, and HBsAg-retention in the endoplasmic reticulum. Rescue of HBeAg expression increased HBsAg-amount but not its release. Expression of the isolated genomes is associated with a higher Nrf2/ARE-dependent gene expression as compared to reference genomes independent of HBeAg expression. Kinome analyses revealed a decreased activity of receptors involved in regulation of proliferative pathways for all patient isolates compared to the reference genomes. No specific conserved mutations could be found between all genomes from Phase 3. HBeAg-negative genomes from Phase 3 exhibit distinct molecular characteristics leading to lower HBsAg synthesis and release, enhanced oxidative stress protection and decreased activity of key kinases, triggering an antiproliferative stage, which might contribute to the lower probability of hepatocellular carcinoma. The observed differences cannot be associated with loss of HBeAg or specific mutations common to all analysed isolates, indicating the phenotype of Phase 3 derived genomes to be the result of a multifactorial process likely reflecting a conserved natural selection process.

Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

Characterization of mutations in hepatitis B virus DNA isolated from Japanese HBsAg-positive blood donors in 2021 and 2022

Missense mutations in certain small envelope proteins diminish the efficacy of antibodies. Consequently, tracking the incidence and types of vaccine-escape mutations (VEMs) was crucial both before and after the introduction of universal hepatitis B vaccination in Japan in 2016. In this study, we isolated hepatitis B virus (HBV) DNA from 58 of 169 hepatitis B surface antigen (HBsAg)-positive blood samples from Japanese blood donors and determined the nucleotide sequence encoding the small envelope protein. DNA from six (10%) of the samples had VEMs, but no missense mutations, such as G145R, were detected. Complete HBV genome sequences were obtained from 29 of the 58 samples; the viral genotype was A1 in one (3%), A2 in three (10%), B1 in nine (31%), B2 in five (17%), B4 in one (3%), and C2 in 10 (34%) samples. Tenofovir-resistance mutations were detected in two (7%) samples. In addition, several core promoter mutations, such as 1762A>T and 1764G>A, and a precore nonsense mutation, 1986G>A, which are risk factors for HBV-related chronic liver disease, were detected. These findings provide a baseline for future research and highlight the importance of ongoing monitoring of VEMs and drug resistance mutations in HBV DNA from HBsAg-positive blood donors without HBV antibodies.

Precore mutation enhances viral replication to facilitate persistent infection especially in HBeAg-negative patients

Naturally occurred precore (PC, G1896A) and/or basal core promoter (BCP, A1762T/G1764A) mutations are prevalent in chronic HBV-infected patients, especially those under HBeAg-negative status. However, the replicative capacity of HBV with PC/BCP mutations remains ambiguous. Herein, meta-analysis showed that, only under HBeAg-negative status, the serum HBV DNA load in patients with PC mutation was 7.41-fold higher than those without the mutation. Both PC mutation alone and BCP ​+ ​PC mutations promoted HBV replication in cell and hydrodynamic injection mouse models. In human hepatocyte chimeric mouse model, BCP ​+ ​PC mutations led to elevated replicative capacity and intrahepatic core protein accumulation. Mechanistically, preC RNA harboring PC mutation could serve as mRNA to express core and P proteins, and such pgRNA-like function favored the maintenance of cccDNA pool under HBeAg-negative status. Additionally, BCP ​+ ​PC mutations induced more extensive and severe human hepatocyte damage as well as activated endoplasmic reticulum stress and TNF signaling pathway in livers of chimeric mice. This study indicates that HBeAg-negative patients should be monitored on HBV mutations regularly and are expected to receive early antiviral treatment to prevent disease progression.

Core Promoter and Pre-Core Variants of the Hepatitis B Virus (HBV) Are Frequent in Chronic Hepatitis B HBeAg-Negative Patients Infected by Genotypes A and D.

In Brazil, hepatitis B virus endemicity is low, moderate, or high in some areas, such as Espírito Santo State in the southeast region. In this study, we intend to characterize the basal core promoter (BCP) and pre-core region (PC) variants and their association with clinical/epidemiological disease patterns in patients infected with genotypes A and D. The study included 116 chronic hepatitis B patients from Espírito Santo State, Southeast Brazil, infected with genotypes A and D. Basal core promoter (BCP) and pre-core mutations were analyzed in these patients. The frequency of BCP and PC mutations was compared with age, HBeAg status, HBV genotype and subgenotype, HBV-DNA level, clinical classification, and transmission route. HBeAg-negative status was found in 101 (87.1%) patients: 87 (75.0%) were infected with genotype A (A1 = 85; A2 = 2) and 29 (25.0%) were infected with genotype D (D3 = 24; D4 = 3; D2 = 2). BCP + PC variants altogether were more frequent (48.1%) in genotype D than in genotype A strains (6.0%) (p < 0.001). When this evaluation was performed considering the cases that presented only the A1762T and/or G1764A (BCP) mutations, it was observed that the frequency was higher in genotype A (67.5%) compared to genotype D (7.4%) (p < 0.001). On the other hand, considering the samples with mutations only in positions G1896A and/or G1899A (PC), the frequency was higher in genotype D (75.8%) than in genotype A (6.9%) (p < 0.001). Interestingly, HBV DNA was lower than 2000 IU/mL especially when both BCP/PC mutations were present (p < 0.001) or when only PC mutations were detected (p = 0.047), reinforcing their role in viral replication.

Detection of Immune Escape and Basal Core Promoter/Precore Gene Mutations in Hepatitis B Virus Isolated from Asymptomatic Hospital Attendees in Two Southwestern States in Nigeria.

Several mutations in the surface (S), basal core promoter (BCP), and precore (PC) genes of the hepatitis B virus have been linked to inaccurate diagnosis and the development of immune escape mutants (IEMs) of the infection, which can lead to chronic infection. Understanding the prevalence and spread of these mutations is critical in the global effort to eliminate HBV. Blood samples were collected from 410 people in Osun and Ekiti states, southwest Nigeria, between 2019 and 2021. Participants were drawn from a group of asymptomatic people who were either blood donors, outpatients, or antenatal patients with no record of HBV infection at the medical outpatients' unit of the hospital. DNA was extracted from plasma using a Qiagen DNEasy kit, followed by nested PCR targeting HBV S and BCP/PC genes. The Sanger sequencing method was used to sequence the positive PCR amplicons, which were further analyzed for IEMs, BCP, and PC mutations. HBV-DNA was detected in 12.4% (51/410) of individuals. After DNA amplification and purification, 47.1% (24) of the S gene and 76.5% (39) of the BCP/PC gene amplicons were successfully sequenced. Phylogenetic analysis showed that all the HBV sequences obtained in this study were classified as HBV genotype E. Mutational analysis of the major hydrophilic region (MHR) and a-determinant domain of S gene sequences revealed the presence of three immune escape mutations: two samples harbored a T116N substitution, six samples had heterogenous D144A/N/S/H substitution, and one sample had a G145E substitution, respectively. The BCP/PC region analysis revealed a preponderance of major BCP mutants, with the prevalence of BCP double substitutions ranging from 38.5% (A1762T) to 43.6% (G1764A). Previously reported classical PC mutant variants were observed in high proportion, including G1896A (33.3%) and G1899A (12.8%) mutations. This study confirms the strong presence of HBV genotype E in Nigeria, the ongoing circulation of HBV IEMs, and a high prevalence of BCP/PC mutants in the cohorts. This has implications for diagnosis and vaccine efficacy for efficient management and control of HBV in the country.

Detection of Mutations in Precore and Basal Core Region in HBeAg Negative Chronic Hepatitis B Patients from Jaipur

Detection of Mutations in Precore and Basal Core Region in HBeAg Negative Chronic Hepatitis B Patients from Jaipur

Clinical isolates of hepatitis B virus genotype C have higher in vitro transmission efficiency than genotype B isolates.

Serum samples were collected from 54 hepatitis B e antigen (HBeAg)-positiveChinese patients infected with hepatitis B virus (HBV)subgenotype B2 or C2. They were compared for transmission efficiency using same volume of samples or infectivity using same genome copy number. Adding polyethylene glycol (PEG)during inoculation did not increase infectivity of fresh samples but markedly increased infectivity following prolonged sample storage. Differentiated HepaRG cells infected without PEG produced more hepatitis B surface antigen (HBsAg)and higher HBsAg/HBeAgratio than sodium taurocholate cotransporting polypeptide (NTCP)-reconstitutedHepG2 cells infected with PEG. They better supported replication of core promoter mutant in contrast to wild-type (WT)virus by HepG2/NTCP cells. Overall, subgenotype C2 samples had higher viral load than B2 samples, and in general produced more HBeAg, HBsAg, and replicative DNA following same-volume inoculation. Precore mutant was more prevalent in subgenotype B2 and had reduced transmission efficiency. When same genome copy number of viral particles was inoculated, viral signals were not necessarily higher for three WT C2 isolates than four WT B2 isolates. Using viral particles generated from cloned HBV genome, three WT C2 isolates showed slightly reduced infectivity than three B2 isolates. In conclusion, subgenotype C2 serum samples had higher transmission efficiency than B2 isolates in association with higher viral load and lower prevalence of precore mutant, but not necessarily higher infectivity. PEG-independent infection by HBV viremic serum samples is probably attributed to a labile host factor.

HBV with precore and basal core promoter mutations exhibits a high replication phenotype and causes ER stress-mediated cell death in humanized liver chimeric mice.

Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.

Hepatitis B Virus Genotypes and Pre-core and Core Genes Mutations in a Sample of Iraqi Patients with Chronic Hepatitis B Infection

Background: Genomic studies of hepatitis B virus (HBV) diversity are becoming increasingly significant to understand how HBV mutations interact with a wide spectrum of clinical and pathological disorders. Objective: This study focused on identifying HBV genotypes and determining the status of pre-core (PC) and core promoter (CP) mutants. Methods: Nested polymerase chain reaction was used to identify the viral genotypes of 100 patients with chronic HBV infection. Only 30 samples out of 100 were selected to determine the prevalence of mutations in the PC and CP by Sanger sequencing. Results: Over 95% of the samples had only D genotype and mixed genotypes 5% (B+C+D) in chronic hepatitis B (CHB) patients. G1898A, G1901A, G1910A and G1915A mutations in PC gene were found in total 15 out of 30 (50%) samples, which were distributed in the following proportions: 12 out of 25 (48%) in patients with hepatitis B e antigen (HBeAg) -ve, 8 samples with mutants at G1898A, G1901A, G1910A and G1915A (four mutations), 4 samples with mutants at G1898A, G 1901A and G1910A (three mutations). In addition, 3 out of 5 (60%) in patients with HBeAg +ve, while no type of any mutation was detected in the core gene in patients with chronic hepatitis type B. Conclusion: The genotype D was predominantly prevalent among HBV in CHB patients with 95% and with 5% in mixed genotypes (B+C+D), while genotype B and C were relatively less prevalent among CHB patients than genotype D. The mutations were found in the PC gene at nucleotides G1898A, G1901A, G1910A and G1915A, while no type of mutation was detected in the core gene. Keywords: Genotypes, pre-core mutation, core mutation, HBV Citation: Hussain HT, Al-Shuwaikh AM, Ahmed AM. Hepatitis B virus genotypes and pre-core and core genes mutations in a sample of Iraqi patients with chronic hepatitis B Infection. Iraqi JMS. 2022; 20(2): 217-225. doi: 10.22578/IJMS.20.2.8

Hepatitis B virus haplotype number at baseline is a predictive marker of functional cure during antiviral therapy for patients with genotypes A and D HBeAg-positive chronic hepatitis B.

We investigated associations between hepatitis B virus (HBV) genome-length haplotype number (HN) at baseline in subjects with HBeAg-positive chronic hepatitis B (CHB), and the likelihood of achieving functional cure during direct-acting antiviral therapy METHOD: We analysed 86 HBeAg-positive baseline samples from patients with HBV genotypes A and D who were enrolled in a Phase II trial of tenofovir disoproxil fumarate (TDF) to determine if HN was a biomarker of HBsAg loss during therapy. Findings were validated using baseline samples from 181 patients with HBV genotypes A and D from an independent clinical trial utilising TDF or tenofovir alafenamide therapy in HBeAg-positive CHB. In the HBeAg-positive test cohort, patients with genotypes A or D and ≤2 haplotypes had a minimum of 21-fold higher likelihood of achieving HBsAg loss on TDF. Baseline HN (p < 0.0001) was a stronger predictor of HBsAg loss on therapy than HBsAg titre (p=0.03), HBeAg titre (p=0.0002), or the presence of HBV basal core promoter (A1762T, p=0.0379 and G1764A, p=0.0176) or G1896A precore mutations (p= 0.0218). This finding was validated in the independent validation cohort. HN was statistically higher in patients with HBV genotypes B or C infection compared to genotypes A and D. Baseline HN ≤2 predicts which patients with HBV genotypes A or D will more likely progress to functional cure on current direct-acting antiviral therapy, with greater accuracy than current biomarkers including baseline HBsAg and HBeAg titre.

A cautionary note to hepatitis B e antigen (HBeAg)-negative test results in pregnant women in an area prevalent of HBeAg-negative chronic hepatitis B.

Maternal hepatitis B e Antigen (HBeAg) positivity poses a risk for hepatitis B virus (HBV) mother-to-child transmission (MTCT). In resource-constrained settings, HBeAg testing is recommended as an alternative to HBV DNA testing to establish antiviral prophylaxis eligibility. Nevertheless, the high prevalence of HBeAg-negative chronic hepatitis B (e-CHB) in many countries should not be overlooked. We studied HBV characteristics and explored the potential MTCT risk among HBeAg-negative/HBsAg-positive expectant mothers in an area prevalent of e-CHB. Among 1348 pregnant mothers screened for HBV infection, 81 (6.0%) were HBsAg-positive. These women were examined for HBeAg, HBV DNA, and cord blood HBV DNA. Sixteen (19.8%) of the HBsAg-positive mothers were HBeAg-positive, whereas 65 (80.2%) were HBeAg-negative, including eight inactive carriers (HBsAg <100 IU/ml, HBV DNA ≤ 2000 IU/ml, and ALT < 40 IU/L). Of the remaining 57 HBeAg-negative mothers, ten revealed HBV Basal Core Promoter or Precore mutations, with three having high viremia (HBV DNA > 200 000 IU/mL), which is associated with a high MTCT risk and therefore qualifies them for antiviral prophylaxis. This pilot study provides a cautionary note to the interpretation of negative HBeAg test results when determining eligibility for MTCT antiviral prophylaxis in situations with limited resources and in regions where e-CHB is prevalent.

Status of HBeAg in association with other baseline tests in Iraqi patients with CHB

Chronic Hepatitis B(CHB) is a serious sequel after Hepatitis B Virus infection, although the rate of chronicity is inverse with age but it may be associated with a significant proportion of deaths related to cirrhosis and liver cancer. HBV serology and measurement of hepatocytic enzymes with viral load can predict disease prognosis and response to treatment. HBeAg refers to a high viral replication and associated with more infectivity to others. This study aimed to determine the impact of HBeAg status in chronically infected patients on various baseline tests. A total of 90 patients with CHB infection who were included in this study. Several standardized measures used for liver function tests, viral load and hepatitis B serological markers. HBeAg was negative in 81/90 (90%) patients and positive in 9/90(10%). There was significant association between HBeAg serostatus and HBV-DNA viral load (p=0.042), as more than half of the cases (55.6%) had high levels of HBV serum DNA (≥2000 IU/ml) and positive HBeAg compared to 24.7% of cases with negative HBeAg. HBeAg seroconversion was not associated with liver function tests, modes of transmission, duration of chronicity, marital status, gender and age. We concluded that HBeAg sero-negativity among the studied cases can predict the inactive or reactive phase of chronic hepatitis B, if the latter is associated with high viral loads, so that further studies are needed to find out associated precore mutants.

Characterization of the tenofovir resistance-associated mutations in the hepatitis B virus isolates across genotypes A to D

Tenofovir is wildly used to treat chronic hepatitis B (CHB) infection due to good potency and high genetic barrier to drug resistance. To date, it remains controversial whether hepatitis B virus (HBV) could be resistant to tenofovir. This study used multiple HBV isolates across genotypes A to D to characterize the reported tenofovir resistance-associated mutations identified from a few clinical case reports. Our data demonstrated that all the rtS78T, rtA194T, and CYEI (S106C, H126Y, D134E, and L269I) mutants exhibited no resistance to tenofovir in vitro. In contrast, the quadruple mutation MLVV (rtL180M, rtT184L, rtA200V, and rtM204V) decreased tenofovir susceptibility, with increased half maximal efficient concentration ranging from 3.28-fold to 5.34-fold, but it severely impaired viral fitness by reducing both replication capacity and infectivity of the mutants. Interestingly, basal core promoter (BCP) mutations A1762T/G1764A and precore (PC) mutation G1896A restored the replication capacity of the MLVV mutant to a level even higher than the wild-type virus without BCP and PC mutations. In conclusion, our data support the role of tenofovir as a first-line agent to treat CHB infection but also indicate tenofovir-resistant mutants could emerge due to multiple mutations accumulated during long-term therapy, particularly in hepatitis B e antigen-negative patients.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity.

Of 350 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk of developing cirrhosis and hepatocellular carcinoma (HCC) later in life. HBV is the most diverse DNA virus, and its genome is composed of four open reading frames: Presurface antigen/surface antigen gene (preS/S), precore/core gene (preC/C), polymerase gene (P), and the X gene (X). HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate. The virus has 10 genotypes (A to J) with different geographical distributions. There are various HBV mutations in the HBV genome, including preC/C mutations, preS/S mutations, P gene mutations, and X gene mutations. The core promoter region plays a vital part in the replication, morphogenesis and pathogenesis of the virus. The precore region also plays a crucial role in viral replication. Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity. preC/C mutations are associated with liver disease progression. Precore mutations stop hepatitis B e antigen (HBeAg) production and basal core promoter mutations downregulate HBeAg production. Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC. The emergence of antiviral-resistant mutations is the main reason for treatment failure. This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity. Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

HBV-RNA, Quantitative HBsAg, Levels of HBV in Peripheral Lymphocytes and HBV Mutation Profiles in Chronic Hepatitis B.

A comprehensive characterization of chronic HBV (CHB) patients is required to guide therapeutic decisions. The cumulative impact of classical and novel biomarkers on the clinical categorization of these patients has not been rigorously assessed. We determined plasma HBV-RNA and HBsAg levels, HBV in peripheral lymphocytes (PBMCs) and HBV mutation profiles in CHB patients. Patient demographics (n = 139) and classical HBV biomarkers were determined during a clinical routine. HBV-RNA in plasma and HBV-DNA in PBMCs were determined by RT-PCR. HBsAg levels were determined using Architect. In samples with HBV-DNA viral load >1000 IU/mL, genotype mutations in precore (PC), basal core promoter (BCP), HBsAg and Pol regions were determined by sequencing. Most patients (n = 126) were HBeAg-negative (HBeAgNeg) with significantly lower levels of HBV-RNA, HBV-DNA and HBsAg compared to HBeAg-positive (HBeAgPos) patients (p < 0.05). HBV genotype D prevailed (61/68), and >95% had BCP/PC mutations. Escape mutations were identified in 22.6% (13/63). HBeAgNeg patients with low levels of HBsAg (log IU ≤ 3) were older and were characterized by undetectable plasma HBV-DNA and undetectable HBV-RNA but not undetectable HBV-DNA in PBMCs compared to those with high HBsAg levels. In >50% of the studied HBeAgNeg patients (66/126), the quantitation of HBsAg and HBV-RNA may impact clinical decisions. In conclusion, the combined assessment of classical and novel serum biomarkers, especially in HBeAgNeg patients, which is the largest group of CHB patients in many regions, may assist in clinical decisions. Prospective studies are required to determine the real-time additive clinical advantage of these biomarkers.

Combining the HBcrAg decline and HBV mutations predicts spontaneous HBeAg seroconversion in chronic hepatitis B patients during the immune clearance phase.

To assess predictive ability of hepatitis B virus (HBV) markers and genome mutations for spontaneous hepatitis B e antigen (HBeAg) seroconversion. A total of 113 chronic hepatitis B (CHB) patients were followed up for 76 weeks without antiviral treatment. Baseline basal core promoter (BCP) and precore mutations were detected and serum hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen (HBcrAg), and HBV DNA levels were serially quantified. Eighteen patients experienced spontaneous HBeAg seroconversion (Group A), and the remaining 95 patients did not experience spontaneous HBeAg seroconversion (Group B). At Week 28, HBsAg (p = 0.03) and HBcrAg (p = 0.01) levels were significantly different between Groups A and B. Reduced HBsAg (p = 0.02) and HBcrAg (p < 0.01) levels from baseline to Week 28 were significantly different between two groups. Multivariate logistic regression showed that lower HBcrAg (odds ratio [OR] = 1.02, p = 0.03) levels at Week 28, and HBcrAg levels with sharp decrease at Week 28 (OR = 0.19, p = 0.02) were related with spontaneous HBeAg seroconversion. The areas under the receiver operating characteristic curve (AUROC) showed that reduction in HBcrAg levels from baseline to Week 28 (0.93, p = 0.001, 95% CI: 0.74-1.08) have excellent prediction value. The mutation frequencies of A1574T (51.11% vs. 18.18%, p = 0.001), G1862A (30.00% vs. 13.03%, p = 0.001), G1896A (27.22% vs. 5.45%, p = 0.001), and C1913G (32.78% vs. 12.73%, p = 0.001) in Group A were significantly higher than Group B. Baseline A1574T, G1862A, G1896A, and C1913G mutations and HBcrAg levels with a sharp decrease at Week 28 were associated with spontaneous HBeAg seroconversion.

Regulatory function of interferon-inducible 44-like for hepatitis B virus covalently closed circular DNA in primary human hepatocytes.

Curing hepatitis B virus (HBV) infection requires elimination of covalently closed circular DNA (cccDNA). Interferon (IFN)-γ has noncytolytic antiviral potential; however, elimination of cccDNA could not be achieved. To enhance the regulatory effect, we comprehensively analyzed the host factors associated with cccDNA amplification and IFN-γ and IFN-α effects using an in vitro HBV infection system showing various transcription levels. Primary human hepatocytes were infected with HBV using genomic plasmids carrying the basic core promoter mutation A1762T/G1764A and/or the precore mutation G1896A and treated with IFN-γ and IFN-α. Comprehensive and functional studies involving microarray and small interfering RNA analysis revealed the host factors related to cccDNA regulation. The HBV infection system reproduced the HBV life cycle and showed various propagation levels. Microarray analysis revealed 53 genes correlated with the cccDNA levels. Of the 53 genes, expression of IFN-induced protein 44-like (IFI44L) was significantly upregulated by IFN-γ and IFN-α. The anti-HBV effect of IFI44L is exerted regardless of IFN-γ or IFN-α by inhibiting the activation of nuclear factor-κB and signal transducer and activator of transcription 1 pathways. Using the in vitro HBV infection system, an IFN-inducible molecule, IFI44L, associated with cccDNA amplification, was identified. These results suggest an innovative molecular strategy for the regulation of HBV cccDNA by controlling a novel host factor, IFI44L.

Precore and Basal Core Promoter Hepatitis B Virus (HBV) Variants Are Present From a Young Age and Differ Across HBV Genotypes.

Hepatitis B virus (HBV) precore (PC) and dual basal core promoter (BCP) mutations halt and down-regulate hepatitis B e antigen (HBeAg) production respectively. PC mutation is rarely associated with HBV genotype A. We sought to examine the association of these variants with HBV genotypes, age, and HBeAg status in a racially diverse population in North America. Prospective study included 1,036 (808 adults, 228 children) participants in the Hepatitis B Research Network. PC and BCP variants were determined by Sanger sequencing, and dominant HBV species (>50%) were reported. Median age was 36.3years (range, 2-80), 44.6% HBeAg(+), 74.2% Asians, 13.3% black, and 9.7% white. The dominant PC variant was present in 29.4% participants, including 20 with subgenotype A1 or A2. Seventeen of 20 participants with genotype A and PC had a compensatory C1858T mutation. In the HBeAg(+) cohort, the prevalence of PC and/or BCP variants increased from 14.4% in the first two decades to 51% after 40years of age. Among those aged 2-18, 52% and 83% with dominant PC and BCP variants were HBeAg(+) compared to 3.8% and 29% in the >40years age group. HBeAg clearance rates were significantly higher for those with dominant PC or BCP variants: 24.4 and 15.0 per 100 person-years compared to 6.0 in wild-type HBV (P<0.0001). PC variants can be present in HBV genotype A and are usually associated with C1858T, which preserves the pregenome encapsidation sequence. Selection of PC and BCP variants occurred at a young age, with increasing prevalence across age groups. HBeAg(+) participants with dominant PC and BCP variants progressed to the HBeAg(-) phase of chronic HBV infection significantly faster. This finding has potential clinical and therapeutic implications.

Epidemiology, Genotyping, Mutational and Phylogenetic Analysis of Hepatitis B Virus Infection in North-east India

Hepatitis B virus (HBV) infection is a major public health problem globally. Northeast India is home to indigenous tribes with different ethnicity and high rates of drug abuse and HIV infection. The study was designed to estimate the burden of HBV infection across various spectrums of liver diseases from this region. HBV genotypes and subgenotypes play a role in the chronicity of disease, response to treatment and its progression. As very limited data are available from this region, we tried to elucidate the role of HBV genotypes, HBV mutants and their phylogenetic analysis. We designed a prospective multicentric study, and included 7464 liver disease cases, 7432 blood donors and 650 health care workers, who were screened for HBV infection. HBV DNA positive patients were genotyped and subjected to surface protein, precore and core mutation and phylogenetic analysis. The prevalence of HBV infection with respect to different types of liver diseases, blood donors and health care workers was 9.9% (1550/15,546). 49.5% (768/1550) cases were found to be HBV DNA positive. The most common genotype was found to be genotype D 74.2% (570/768), followed by genotype C 6.5% (50/768), A 4.4% (34/768) and I 0.9% (7/768). This study highlights the high hepatitis B burden in Northeast India, reflecting lacunae in health care needs of the region. Also, the different genotype distribution and presence of mutationsmay translate into different rates of liver disease progression, prognosis and ultimately, clinical significance. However, further prospective cohort study from Northeast India is warranted, to elucidate the clinical significance of multiple genotypes and mutation in this unique population.