Core promoter: A critical region where the hepatitis B virus makes decisions

The core promoter(CP) of the viral genome plays an important role for hepatitis B virus(HBV) replication as it directs initiation of transcription for the synthesis of both the precore and pregenomic(pg) RNAs. The CP consists of the upper regulatory region and the basa core promoter(BCP). The CP overlaps with the 3'-end of the X open reading frames and the 5'-end of the precore region,and contains cis-acting elements that can independently direct transcription of the precore mRNA and pgRNA. Its transcription regulation is under strict control of viral and cellular factors. Even though this regulatory region exhibits high sequence conservation,when variations appear,they may contribute to the persistence of HBV within the host,leading to chronic infection and cirrhosis,and eventually,hepatocellular carcinoma. Among CP sequence variations,those occurring at BCP may dysregulate viral gene expression with emphasis in the hepatitis B e antigen,and contribute to disease progression. In this review these molecular aspects and pathologic topics of core promoter are deeply evaluated.

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 theχgene(χ).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,andχgene 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.

Precore/basal core promoter mutants and hepatitis B viral DNA levels as predictors for liver deaths and hepatocellular carcinoma

AIM： To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METHODS： The mean follow-up time was 83.6 ± 39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA （HBV DNA） level and hepatitis B e antigen （HBeAg） were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development. RESULTS： During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio： 95.74 （12.13-891.31）, P 〈 0.0001], male sex [odds ratio： 7.61 （2.20-47.95）; P = 0.006], and higher Iogzo HBV DNA [odds ratio： 4.69 （1.16-20.43）; P 〈 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio： 26.51 （2.36-381.47）; P = 0.007], presence of precore mutants [odds ratio： 4.23 （1.53-19.58）, P = 0.02] and presence of basal core promoter mutants [odds ratio： 2.93 （1.24-7.57）; P = 0.02] were independent predictors for progression to hepatocellular carcinoma. CONCLUSION： Our results show that high levels of baseline serum HBV DNA are associated with non- hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.

Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients

AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ2 = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ2 = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia

AIM： To identify the distribution of hepatitis B virus （HBV） subgenotype and basal core promoter （BCP） mutations among patients with HBV-associated liver disease in Indonesia.METHODS： Patients with chronic hepatitis （CH, n =61）, liver cirrhosis （LC, n = 62）, and hepatocellular carcinoma （HCC, n = 48） were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing.RESULTS： HBV genotype B （subgenotypes B2, B3, B4, 85 and B7） the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C （subgenotypes C1, C2 and C3） was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 （84.9%） and C1 （82.2%） were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 （84.9%） and adrq＋ （89.4%） were the most prevalent in HBV genotype B and C, respectively. Double mutation （A1762T/G1764A） in the BCP was significantly higher in LC （59.7%） and HCC （54.2%） than in CH （19.7%）, suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC （46.8%）, but lower in HCC （22.9%） and CH （18.0%）, suggesting that this mutation may be an indicator of cirrhosis.CONCLUSION： HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.

Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients

AIM:To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients.METHODS:Sixty-four patients with chronic hepatitis,65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study.HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing.Viral load was measured by real-time polymerase chain reaction.RESULTS:Of 179 patients,108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted.The A1896 mutation was not found in HBeAg(+) patients,however,this mutation was detected in 70.7% of HBeAg(-) patients.This mutation was frequently found when HBeAg was not expressed (87.7%),compared to that found in HBeAg seroconverted patients (65.1%).The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P=0.004).The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients,however,the prevalence of this mutation did not significantly differ among the two groups (P=0.054).In HBeAg(+) patients,the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001).The A1899 mutation did not correlate with HBV DNA (P=0.609).In HBeAg(-) patients,the T1762/A1764 mutation alone was not correlated with HBV DNA (P=0.095),however,the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001).CONCLUSION:The percentage of HBeAg(-) patients is high in Indonesia,and most of the HBeAg(-) patients had been seroconverted.The A1896 mutation was most likely the major cause of HBeAg loss.The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients,but not in HBeAg(-) patients.

Relationship between the different replication status of HBV and mutations in the core promoter in mothers and their children infected via mother-to-infant transmission

OBJECTIVE: To study the relationship between the different replication status of hepatitis B virus (HBV) and mutations in the core promoter (CP) in mother and her child infected by mother-to-infant transmission. METHODS: The core promoter was amplified by PCR and cloned into pGEM-T vector with the T-A choning technique. The recombinant plasmid pGEM-CP was confirmed by digestion with restriction enzyme Apa I and Sac I. Two clones were selected to be sequenced in each patient. RESULTS: Every pair of mother and child had same serotype and genotype and the homology of nucleotides encoding 'a' determinant was 98%-100%. The number of mutations in the core promoter of patients with a high replication status was less than that in those with a low replication status. Mutations were mainly distributed in basia core promoter (BCP) and the inbibitor region of Kunitz-type serine protease. This difference was not associated with mother or child. CONCLUSION: The different replication status of HBV is caused by mutations in the core promoter in mother and child infected hy mother-to-infant transmission and appears to be not associated with the status of development of the infection.

Quasispecies groups in the core promoter region of hepatitis B virus

Objectives: To investigate the mutation of the basic core promoter (BCP) of hepatitis B virus (HBV) and clarify the significance of HBV quasispecies groups in patients with chronic HBV infection. Methods: A set of specific primers was synthesized according to the HBV DNA sequence of a Chinese strain. The BCP was amplified by PCR method from the serum of 40 patients with chronic HBV infection, and the PCR products of 2 patients were subcloned into pGEM Teasy vectors. Polyacrylamide gel elec- trophoresis (PAGE) was employed to display the de- letion mutations, and clones with differential length were selected to be sequenced. Sequence comparison was made to find the difference. Results: Two or three bands were displayed by PAGE in 60% patients. The results of sequence anal- ysis showed that there are some kinds of mutations in the BCP region. The substitution always occurs in TATA-like boxes, especially from T to C on 140 site. The deletion mutations were detected in TA1, TA2 and TA3. The 8bp, 20bp deletion mutations fre- quently happened. Conclusions: There is a hot deletion region in the BCP. The deletion and the substitution in the TATA- like box may influence the expression of preC/C pro- tein. The sequencing results indicate that there are HBV quasispecies groups in patients with chronic HBV infection.

Developing a CRISPR/FrCas9 system for core promoter editing in rice

Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths.However,targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s.In this study,we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing.Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM.In addition,FrCas9 demonstrated activity against all 16 possible NNTA PAMs,achieving an efficiency of up to 35.3%in calli and generating homozygous or biallelic mutations in 31.3%of the T_(0)transgenic plants.A proof-ofconcept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content.Multiplex mutations and deletions were produced by bidirectional editing,mediated by FrCas9,using a single palindromic TATA sequence as a PAM.Moreover,we developed FrCas9-derived base editors capable of programmable conversion between AT and GC pairs in plants.This study highlights a versatile FrCas9 toolset for plant core promoter editing,offering great potential for the fine-tuning of gene expression and creating of new germplasms.

Etiological roles of core promoter variation in triple-negative breast cancer

Abnormal geneexpression playskeyrole in cancerdevelopment.Acorepromoter is located around the transcriptional start site.Through interaction between core promoter sequences and transcriptional factors,core promoter controls transcriptional initiation.We hypothesized that in cancer,core promoter sequences could be mutated to interfere the interaction with transcriptional factors,resulting in altered transcriptional initiation and abnormal gene expression and cancer development.We used triple-negative breast cancer(TNBC)as a model to test our hypothesis.We collected genome-wide core promoter variants from 279 TNBC genomes.After extensive filtering of normal genomic polymorphism,we identified 19,427 recurrent somatic variants in 1,238 core promoters of 1,274 genes and 1,694 recurrent germline variants in 272 core promoters of 294 genes.Many of the affected genes were oncogenes and tumor suppressors.Analysis of RNA-seq data from the same patient cohort identified increased or decreased gene expression in 439 somatic and 85 germline variantsaffected genes,and the results were validated by luciferase reporter assay.By comparing with the core promoter variation data from 610 unclassified breast cancer,we observed that core promoter variants in TNBC were highly TNBC-specific.We further identified the drugs targeting the genes with core promoter variation.Our study demonstrates that core promoter is highly mutable in cancer,and can play etiological roles in TNBC and other types of cancer through influencing transcriptional initiation.

Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells

Complement receptor 2 （CR2/CD21） is predominantly expressed on the surface of mature B cells where it forms part of a coreceptor complex that functions, in part, to modulate B-cell receptor signal strength. CR2/CD21 expression is tightly regulated throughout B-cell development such that CR2/CD21 cannot be detected on pre-B or terminally differentiated plasma cells. CR2/CD21 expression is upregulated at B-cell maturation and can be induced by IL-4 and CD40 signaling pathways. We have previously characterized elements in the proximal promoter and first intron of CR2/CD21 that are involved in regulating basal and tissue-specific expression. We now extend these analyses to the CR2/CD21 core promoter. We show that in mature B cells, CR2/~D21 transcription proceeds from a focused TSS regulated by a non-consensus TATA box, an initiator element and a downstream promoter element. Furthermore, occupancy of the general transcriptional machinery in pre-B versus mature B-cell lines correlate with CR2/CD21 expression level and indicate that promoter accessibility must switch from inactive to active during the transitional B-cell window.

Conserved Curvature of RNA Polymerase Ⅰ Core Promoter Beyond rRNA Genes: The Case of the Tritryps

In trypanosomatids, the RNA polymerase I （RNAPI）-dependent promoters controlling the ribosomal RNA （rRNA） genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no con- formational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leiskmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such con- served curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.

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 BCPþPC mutations promoted HBV replication in cell and hydrodynamic injection mouse models.In human hepatocyte chimeric mouse model,BCPþPC 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,BCPþPC 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.

Systematic evaluation of HBV BCP/PC mutations on the risk of hepatocarcinogenesis

Objective:To evaluate of the effects of mutations in BCP-A1762T/G1764A and PC-G1896A genes on hepatocarcinogenesis.Methods:Computer searches for PubMed,SCI,CNKI,VIP and WanFang Data databases were conducted to collect literature on the role of mutations in the disease process associated with HBV infection from database creation to July 1,2021.Two researchers independently screened the articles,extracted information and evaluated the quality of the studies.Review Manager software version 5.4 was used for Meta-analysis.Results:A total of 40 articles were included,with a total of 12423 cases and 3710 cases of hepatocellular carcinoma.Meta-analysis showed that mutations in BCP-A1762T/G1764A gene were associated with the disease process of HBV infection and promoted hepatocellular carcinogenesis.mutations in BCP/PC gene were significant in the process of HBV infection in BCP-A1762T/G1764A in HCC vs non-HCC[OR=4.05,95%CI=2.64~6.22],CHBC[OR=3.90,95%CI=2.13~7.17],CHB[OR=2.77,95%CI=1.78~4.32],LC[OR=1.64,95%CI=0.95~2.84],which were statistically significant;in PC-G1896A mutation HCC vs non-HCC[OR=1.49,95%CI=1.02~2.17],CHBC[OR=1.56,95%CI=0.89~2.72],CHB[OR=1.80,95%CI=1.17~2.77]were statistically significant,while the difference was not statistically significant when comparing HCC with LC(P=0.4).The BCP-A1762T/G1764A mutation in the B genotypes/genotyped versus the C genotype[OR=0.36,95%CI=0.20~0.64],with a statistically significant difference,and no statistically significant difference in the PC-G1896A mutation.BCP-A1762T/G1764A mutation in the C gene in HCC versus non-HCC[OR=3.71,95%CI=1.82~7.61]and PC-G1896A mutation in HCC vs non-HCC[OR=2.81,95%CI=1.34~5.91],the differences were statistically significant.Conclusions:Current evidence suggests that mutations in the BCP-A1762T/G1764A and PC-G1896A genes have a significant effect on the increased risk of hepatocellular carcinoma and are genotype dependent.However,due to the limitation of the number and quality of included studies,these findings need to be validated by more high-quality studies.

Hepatitis B virus core protein as a Rab-GAP suppressor driving liver disease progression

Chronic hepatitis B virus(HBV)infection can lead to advanced liver pathology.Here,we establish a transgenic murine model expressing a basic core promoter(BCP)-mutated HBV genome.Unlike previous studies on the wild-type virus,the BCP-mutated HBV transgenic mice manifest chronic liver injury that culminates in cirrhosis and tumor development with age.Notably,agonistic anti-Fas treatment induces fulminant hepatitis in these mice even at a negligible dose.As the BCP mutant exhibits a striking increase in HBV core protein(HBc)expression,we posit that HBc is actively involved in hepatocellular injury.Accordingly,HBc interferes with Fis1-stimulated mitochondrial recruitment of Tre-2/Bub2/Cdc16 domain family member 15(TBC1D15).HBc may also inhibit multiple Rab GTPase-activating proteins,including Rab7-specific TBC1D15 and TBC1D5,by binding to their conserved catalytic domain.In cells under mitochondrial stress,HBc thus perturbs mitochondrial dynamics and prevents the recycling of damaged mitochondria.Moreover,sustained HBc expression causes lysosomal consumption via Rab7 hyperactivation,which further hampers late-stage autophagy and substantially increases apoptotic cell death.Finally,we show that adenovirally expressed HBc in a mouse model is directly cytopathic and causes profound liver injury,independent of antigen-specific immune clearance.These findings reveal an unexpected cytopathic role of HBc,making it a pivotal target for HBV-associated liver disease treatment.The BCP-mutated HBV transgenic mice also provide a valuable model for understanding chronic hepatitis B progression and for the assessment of therapeutic strategies.

Gene heterogeneity of hepatitis B virus isolates from patients with severe hepatitis B

BACKGROUND: The pathogenesis of severe hepatitis B remains unknown. Reports have indicated that hepatitis B virus (HBV) mutations are important factors in the pathogenesis of this disease. This study was to investigate the genetic heterogeneity of HBV strains from serum samples of patients with fulminant hepatitis B. METHODS: Full-length HBV genomes from 4 patients with severe hepatitis B were cloned and sequenced to observe mutations in every open reading-frame ( ORF). Serum samples of another 25 patients with severe hepatitis B, 30 patients with chronic hepatitis B, and 25 HBV carriers were collected for sequencing and comparison of mutations in preS2, preC and core promoter regions. RESULTS: Of 4 HBV full-length genome sequences, 3 had a G to A mutation at nucleotide A1896 in the preC region and 1 had double mutations of T1762-A1764 in the core promoter region. The 4 sequences showed mutations in the known B or T cell epitopes of the preS2 and C regions. For the other 3 groups, more mutations were seen in the preS2 region in the HBV isolates from the patients with severe hepatitis B than those from the patients with chronic hepatitis B and HBV carriers (P <0.01). There was a significant difference of mutations in the T cell epitope region of preS2 between the patients with severe hepatitis B and those with chronic hepatitis B or HBV carriers (P <0.01). In the preC and core promoter regions, the mutation frequencies of T1653 and C1753 were 48.0% and 24.0% respectively in the patients with severe hepatitis B, but none of these mutations were observed in the patients with chronic hepatitis B group or HBV carriers (P <0.01). The mutation frequency of T1762-A1764 was 76.0% in the patients with severe hepatitis B, 40.0% in the patients with chronic hepatitis B (P <0. 01) , and 16. 0% in the HBV carriers ( P < 0. 01). There was a significant difference in A1896 mutation between the patients with severe hepatitis B and the patients with chronic hepatitis B (P < 0. 05 ) or the HBV carriers (P<0.05). CONCLUSION: Our observations suggest that the accumulation and persistence of high frequency mutations or complex mutations may be associated with the development and deterioration of HBV infection.

HBeAg negative variants and their role in the natural history of chronic hepatitis B virus infection

Molecular virology methods including polymerase chain reaction, cloning and sequencing have revolutionised our understanding of viral genome variation. In the case of hepatitis B virus (HBV), sequencing studies have identified a number of virus variants normally found during the natural course of chronic infection. The appearance of the precore stop codon (with G-for-A substitution at position 1896) and basal core promoter (BCP) (with A-for-T and G-for-A, at positions 1762 and 1764, respectively) variants which reduce or abrogate hepatitis B e antigen (HBeAg) production, heralds the initiation of the seroconversion phase from HBeAg to anti-HBe positivity. The gradual removal of the tolerogenic effect of HBeAg leads to the awakening of the immune response (immune clearance phase). Most patients after HBeAg seroconversion become &#x0201c;inactive HBsAg carriers&#x0201d;. However during the course of infection precore and/or BCP variants may emerge and be selected leading to HBeAg negative chronic hepatitis B (CHB) with high viremia levels (reactivation phase). The prevalence of HBeAg negative CHB has been increasing over the last few decades and has become the commonest type of HBV infection in many countries of the world. This probably reflects the aging of existing HBV carriers and the effective prevention measures restricting new HBV infections. Frequent acute exacerbations accompanied by high viral replication, elevated alanine aminotransferase levels and histological activity are a common feature of HBeAg negative CHB leading to cirrhosis much faster than in HBeAg positive CHB patients.

Quantitation of HBsAg predicts response to entecavir therapy in HBV genotype C patients

AIM： To analysis the factors that predict the response to entecavir therapy in chronic hepatitis patients with hepatitis B virus （HBV） genotype C. METHODS： Fifty patients [hepatitis B e antigen （HBeAg）- negative：HBeAg-positive = 26：24] with HBV genotype C, who received nalve entecavir therapy for 〉 2 years, were analyzed. Patients who showed HBV DNA levels ≥ 3.0 log viral copies/mL after 2 years of entecavir ther- apy were designated as slow-responders, while those that showed 〈 3.0 log copies/mL were termed rapid- responders. Quantitative hepatitis B surface antigen （HBsAg） levels （qHBsAg） were determined by the Archi- tect HBsAg QT immunoassay. Hepatitis B core-related antigen was detected by enzyme immunoassay. Pre-C and Core promoter mutations were determined using by polymerase chain reaction （PCR）. Drug-resistance muta- tions were detected by the PCR-Invader method. RESULTS： At year 2, HBV DNA levels in all patients in the HBeAg-negative group were 〈 3.0 log copies/mL. In contrast, in the HBeAg-positive group, 41.7% were slow-responders, while 58.3% were rapid-responders. No entecavir-resistant mutants were detected in the slow-responders. When the pretreatment factors were compared between the slow- and rapid-responders; the median qHBsAg in the slow-responders was 4.57 log IU/mL, compared with 3.63 log IU/mL in the rapid- responders （P 〈 0.01）. When the pretreatment factors predictive of HBV DNA-negative status at year 2 in all 50 patients were analyzed, HBeAg-negative status, low HBV DNA levels, and low qHBsAg levels were signifi- cant （P 〈 0.01）. Multivariate analysis revealed that the low qHBsAg level was the most significant predictive factor （P = 0.03）. CONCLUSION： Quantitation of HBsAg could be a use- ful indicator to predict response to entecavir therapy.

Characterization of hepatitis B virus genotypes/subgenotypes in 1301 patients with chronic hepatitis B in North China

Background There is still a paucity of data on hepatitis B virus （HBV） subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen （HBeAg）-Ioss-associated mutations in patients with chronic hepatitis B （CHB） is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence. Methods Sera were collected from 1301 nucleos（t）ide analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter （BCP） regions. Results HBV/B, HBV/C, and HBV/D were detected in 190 （14.6%）, 1096 （84.2%）, and 15 （1.2%） patients, respectively. HBV/B2 （182/190）, HBV/C2 （1069/1096）, and HBV/D1 （12/15） were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area （77.2%） than in other north areas （84.9%-87.4%）. HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase （ALT） levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM2041N （44.9% vs. 30.2%, P 〈0.01） and BCP mutations A1762T＋G1764A （65.8% vs. 40.0%, P〈0.01） compared with HBV/B infection. Conclusions C2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection.