Sex disparity in viral load, inflammation and liver damage in transgenic mice carrying full hepatitis B virus genome with the W4P mutation in the preS1 region

AIM To study sex disparity in susceptibility to hepatocellular carcinoma(HCC), we created a transgenic mouse model that expressed the full hepatitis B virus(HBV) genome with the W4P mutation.METHODS Transgenic mice were generated by transferring the p HY92-1.1 x-HBV-full genome plasmid(genotype A2) into C57 Bl/6 N mice. We compared serum levels of hepatitis B surface antigen(HBs Ag), interleukin(IL)-6, and the liver enzymes alanine aminotransferase(ALT) and aspartate transaminase(AST), as well as liver histopathological features in male and female transgenic(W4PTG) mice and in nontransgenic littermates of 10 mo of age. RESULTS W4PTG males exhibited more pronounced hepatomegaly, significantly increased granule generation in liver tissue, elevated HBs Ag expression in the liver and serum, and higher serum ALT and IL-6 levels compared to W4PTG females or littermate control groups. CONCLUSION Together, our data indicate that the W4 P mutation in pre S1 may contribute to sex disparity in susceptibility to HCC by causing increased HBV virion replication and enhanced IL-6-mediated inflammation in male individuals. Additionally, our transgenic mouse model that expresses full HBV genome with the W4 P mutation in pre S1 could be effectively used for the studies of the progression of liver diseases, including HCC.

Analysis of hepatitis B virus preS1 variability and prevalence of the rs2296651 polymorphism in a Spanish population

AIM To determine the variability/conservation of the domain of hepatitis B virus(HBV) pre S1 region that interacts with sodium-taurocholate cotransporting polypeptide(hereafter, NTCP-interacting domain) and the prevalence of the rs2296651 polymorphism(S267 F, NTCP variant) in a Spanish population. METHODS Serum samples from 246 individuals were included and divided into 3 groups: patients with chronic HBV infection(CHB)(n = 41, 73% Caucasians), patients with resolved HBV infection(n = 100, 100% Caucasians) and an HBV-uninfected control group(n = 105, 100% Caucasians). Variability/conservation of the amino acid(aa) sequences of the NTCPinteracting domain,(aa 2-48 in viral genotype D) and a highly conserved pre S1 domain associated with virion morphogenesis(aa 92-103 in viral genotype D) were analyzed by next-generation sequencing and compared in 18 CHB patients with viremia > 4 log IU/mL. The rs2296651 polymorphism was determined in all individuals in all 3 groups using an in-house real-time PCR melting curve analysis.RESULTS The HBV pre S1 NTCP-interacting domain showed a high degree of conservation among the examined viral genomes especially between aa 9 and 21(in the genotype D consensus sequence). As compared with the virion morphogenesis domain, the NTCPinteracting domain had a smaller proportion of HBV genotype-unrelated changes comprising > 1% of the quasispecies(25.5% vs 31.8%), but a larger proportion of genotype-associated viral polymorphisms(34% vs 27.3%), according to consensus sequences from Gen Bank patterns of HBV genotypes A to H. Variation/conservation in both domains depended on viral genotype, with genotype C being the most highly conserved and genotype E the most variable(limited finding, only 2 genotype E included). Of note, proline residues were highly conserved in both domains, and serine residues showed changes only to threonine or tyrosine in the virion morphogenesis domain. The rs2296651 polymorphism was not detected in any participant.CONCLUSION In our CHB population, the NTCP-interacting domain was highly conserved, particularly the proline residues and essential amino acids related with the NTCP interaction, and the prevalence of rs2296651 was low/null.

Cloning and expression of the preS1 gene of hepatitis B virus in yeast cells

Objective: To investigate the complex functions of HBV preS1 protein, we constructed HBV preS1 gene expression vector and expressed it in yeast cells. Methods: Polymerase chain reaction (PCR) was per- formed to amplify the gene of HBV preS1 from the plasmid pCP10 containing the whole DNA fragment of HBV ayw subtype as template and the PCR prod- uct was cloned into the pGEM-T vector for sequen- cing. After being identified, the HBV preSl gene was cut from the pGEM-T vector by EcoR I and Pst I restriction enzymes, and cloned into yeast expres- sive plasmid pGBKT7 to constructe pGBKT7-preS1 recombinant expressive plasmid. This plasmid was transformed into yeast cell AH109 and expressed in it. The yeast protein was isolated and analyzed with sodium dodecyl suifate-polyacrylamide gel electro- phoresis(SDS-PAGE) and Western blotting. Results: The HBV preS1 gene was amplified success- fully and identified by DNA sequencing. The PCR products were coincided completely with the reported sequence. The digested fragments were cloned into the pGBKT7 vector and transformed into yeast cell AH109. The results of SDS-PAGE and Western blot- ting assay showed: (1) The HBV preS1 protein was expressed and existed in yeast cells; (2) The molecu- lar weight of the expression product was about 30 000 D. Conclusion: The HBV preS1 gene was successfully cloned and expressed in yeast cells.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preSl (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preSl (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

血清preS1水平与慢性乙型肝炎患者肝纤维化的关系及对癌变的诊断价值

目的分析血清乙型肝炎病毒(hepatitis B virus,HBV)前S1蛋白(precursor S1 protein,preS1)与慢性乙型肝炎(chronic hepatitis B,CHB)肝纤维化及癌变进展的相关性。方法对2019年10月—2021年10月期间在青海红十字医院接受检查的228例乙肝表面抗原(hepatitis B surface antigen,HBsAg)阳性慢性HBV感染者进行回顾性分析,其中CHB患者75例、肝硬化(liver cirrhosis,LC)患者93例(LC组)、肝细胞癌(hepatocellular carcinoma,HCC)患者60例(HCC组)。根据LC和HCC组肝组织活检分析肝脏炎症活动及肝纤维化程度。结果HCC组血清preS1水平[496.32(457.63,988.0)ng/mL]和LC组[338.72(247.93,554.61)ng/mL]血清preS1水平均显著高于CHB组[113.69(87.09,177.40)ng/mL],且差异具有统计学意义(P均<0.05)。HCC组血清preS1水平亦高于LC组(P=0.002)。经受试者工作特征曲线分析,血清preS1水平鉴别诊断CHB与LC的曲线下面积(area under the curve,AUC)是0.881(95%CI:0.830~0.932),鉴别诊断CHB/LC与HCC的AUC是0.861(95%CI:0.815~0.908)。3组患者的血清preS1水平与HBsAg(rs=0.799,P<0.001)呈强正相关和Log HBV DNA(rs=0.262,P<0.001)呈弱正相关。此外LC组和HCC组血清preS1水平与肝脏炎症活动分级(rs=0.201,P=0.009)及肝纤维化分期也呈弱正相关性(rs=0.295,P<0.001)。结论血清preS1水平与血清HBsAg、HBV DNA水平和肝脏炎症和纤维化进展呈正相关,有可能成为鉴别诊断HBV相关慢性肝病肝硬化或癌变的候选标志物。

HMGB1 gene polymorphisms in patients with chronic hepatitis B virus infection

AIM: To characterize high mobility group box chromosomal protein 1(HMGB1) polymorphisms in patients infected with hepatitis B virus(HBV) and determine the different patterns in patient subgroups.METHODS: A total of 1495 unrelated Han Chinese HBV carriers were recruited in this hospital-based casecontrol study.The HMGB1 1176 G/C polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay.RESULTS: A significant association was observed between HMGB1 1176 G/C polymorphism and outcome of HBV infection.The subjects bearing 1176G/G genotype had an increased risk of susceptibility to chronic hepatitis B,liver cirrhosis and severe hepatitis B when compared with those bearing at least one 1176C allele.CONCLUSION: Patients with 1176G/G genotype of HMGB1 gene are more likely to have a progressive status in HBV infection.

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.

Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma

Hepatocellular carcinoma(HCC), the predominant type of primary liver cancer, is one of the most serious lifethreatening malignancies, worldwide. In majority of the cases, HCC develops after prolonged and persistent chronic liver disease. hepatitis B virus(HBV) or HCV infection is prominent etiological factors, attributing to this condition. It has been well documented that HBV, being the inducer of chronic inflammation, is the main causative agent in causing HCC, particularly in Asian countries. The HBV infection leads to a wide range of clinical symptoms from carrier state to malignancy. Cytokines being immune-modulatory molecules, are the key mediators in the defense mechanism against viral infection. In this regard, this review will detail the substantial role of key Th1: interleukin 1(IL-1), IL-2, IL-12, tumor necrosis factor-α, interferon-γ; Th2: IL-4, IL-10 and non Th1/Th2: IL-6, transforming growth factor-β1 cytokines genotypes in analyzing the variability in the clinical manifestations in an HBV-afflicted individual, which might finally, culminates into HCC. Since cytokine production is regulated genetically, the cytokine promoter region single-nucleotide polymorphisms induced changes, greatly affects the cytokine production, thus resulting into differential outcome of immune balance.

Potential mechanisms of hepatitis B virus induced liver injury

Chronic active hepatitis(CAH) is acknowledged as an imperative risk factor for the development of liver injury and hepatocellular carcinoma.The histological end points of CAH are chronic inflammation,fibrosis and cirrhosis which are coupled with increased DNA synthesis in cirrhotic vs healthy normal livers.The potential mechanism involved in CAH includes a combination of processes leading to liver cell necrosis,inflammation and cytokine production and liver scaring(fibrosis).The severity of liver damage is regulated by Hepatitis B virus genotypes and viral components.The viral and cellular factors that contribute to liver injury are discussed in this article.Liver injury caused by the viral infection affects many cellular processes such as cell signaling,apoptosis,transcription,DNA repair which in turn induce radical effects on cell survival,growth,transformation and maintenance.The consequence of such perturbations is resulted in the alteration of bile secretion,gluconeogenesis,glycolysis,detoxification and metabolism of carbohydrates,proteins,fat and balance of nutrients.The identification and elucidation of the molecular pathways perturbed by the viral proteins are important in order to design effective strategy to minimize and/or restore the hepatocytes injury.

Integrative analysis of aberrant Wnt signaling in hepatitis B virus-related hepatocellular carcinoma

AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma(HCC).METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer(COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function(GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of Cp G islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nestedreverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ2 test as appropriate.RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC(P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies(P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates thatthe mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC.CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.

Expression and purification of the complete PreS region of hepatitis B Virus

AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.

Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma

Hepatitis B virus X protein(HBx) plays an important role in the development of hepatocellular carcinoma(HCC). In addition, hepatoma upregulated protein(HURP) is a cellular oncogene that is upregulated in a majority of HCC cases. We highlight here recent findings demonstrating a link between HBx, HURP and anti-apoptosis effects observed in cisplatin-treated HCC cells. We observed that Hep3B cells overexpressing HBx display increased HURP mRNA and protein levels, and show resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reverses this effect, and sensitizes cells to cisplatin. The anti-apoptotic effect of HBx requires activation of the p38/MAPK pathway as well as expression of SATB1, survivin and HURP. Furthermore, silencing of HURP using short-hairpin RNA promotes accumulation of p53 and reduces cell proliferation in SK-Hep-1 cells(p53^(+/–)), whereas these effects are not observed in p53-mutant Mahlavu cells. Similarly, HURP silencing does not affect the proliferation of H1299 lung carcinoma cells or Hep3 B HCC cells which lack p53. Silencing of HURP sensitizes SK-Hep-1 cells to cisplatin. While HURP overexpression promotes p53 ubiquitination and degradation by the proteasome, HURP silencing reverses these effects. Inoculation of SK-Hep-1 cancer cells in which HURP has been silenced produces smaller tumors than control in nude mice. Besides, gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, is upregulated following HURP expression, and silencing of gankyrin reduces HURP-mediated downregulation of p53. In addition, we observed a positive correlation between HURP and gankyrin protein levels in HCC patients(r^2 = 0.778; n = 9). These findings suggest a role for the viral protein HBx and the host protein HURP in preventing p53-mediated apoptosis during cancer progression and establishment of chemoresistance.

mi R-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.

Hydrophobicity of reactive site loop of SCCA1 affects its binding to hepatitis B virus

AIM: To investigate the role of SCCA2 and other SCCA1 molecules in the process of hepatitis B virus (HBV) binding to mammalian cells. METHODS: SCCA1 and SCCA2 were isolated from HepG2. Binding protein (BP) genes were obtained through PCR. Recombinant baculoviruses expressing SCCA1, SCCA2, BP, and different mutants were constructed and utilized to infect mammalian cells to investigate the binding ability of infected cells to HBV. RESULTS: A SCCA1 gene (A1) was isolated from HepG2, but it appeared to lack the binding ability of infected cells to HBV. Two mutants, A1-BP and BP-A1, were constructed by interchanging the carboxyl terminal of A1 and BP. Cells expressing A1-BP showed an increased virus binding capacity, but not BP-A1. Comparison of A1 sequence with the sequence of BP indicated the presence of only three amino acid changes in the carboxyl terminal, two of them were found in the reactive site loop (RSL) of SCCA1. Primary structure assay revealed that the hydrophobicity of BP and AJ515706 in this domain was strong, but A1 was relatively weak. Changing the aa349 of A1 from low hydrophobic glutamic add to high hydrophobic valine enhanced HBV binding. In contrast, HBV binding was reduced by changing the aa349 of BP from valine to glutamic acid. CONCLUSION: The results suggest that the hydrophobicity of RSL of SCCA1 may play an important role in HBV binding to cells.

IFIT1 polymorphisms predict interferon-α treatment efficiency for hepatitis B virus infection

AIM To investigate the association between interferoninduced protein with tetratricopeptide repeats 1(IFIT1) polymorphisms and interferon-α(IFNα) treatment efficiency among Chinese hepatitis B virus(HBV) infection patients.METHODS Two hundred and twenty five newly diagnosed chronichepatitis B(CHB) patients were enrolled in the study. All of these patients received IFNα treatment for a course of 48 wk, and were followed up for 24 wk after the treatment was end. Clinical information about virological response, hepatitis B e antigen(HBe Ag) seroconversion rate and combined response at the end of the treatment, as well as the sustained response by the time of following up 24 wk after the treatment, was collected. Four tag-single nucleotide polymorphisms(SNPs) of IFIT1 were selected and assessed for their association with these clinical outcomes.RESULTS At the end of the treatment, HBe Ag seroconversion was observed in 27.1% patients. Thirty-six point nine percent patients achieved virological response, and 15.6% patients exhibited combined response. Sustained response was obtained in 26.2% patients. The main HBV genotype of the study was genotype B. Patients who infected with HBV genotype B or C showed better treatment efficiency, no matter which clinical outcome was considered. Among the four SNPs assessed, rs303218(A > G) was found to be significantly associated with the end point virological response when assuming additive model [OR = 0.64(95%CI: 0.42-0.96), P = 0.032]. Patients who carried rs303218 GG genotype had a rather higher rate of achieving virological response(response rate: 52%, OR = 0.40, 95%CI: 0.18-0.91; P = 0.028) when compared to those had AA genotype(response rate: 27%). The most significant interaction was observed in patients who had relative lower baseline aspartate transaminase. No association between SNPs and HBe Ag seroconversion, combined response or sustained response was observed.CONCLUSION IFIT1 involves in the regulation of IFNα treatment for CHB and its polymorphism rs303218 can predict the end point virological response. The finding requires further validation.

The influence of hepatitis B virus X protein on the clock genes in liver cells and its significance

Objective： The aim of this study was to investigate the influence of hepatitis B virus X protein （HBx） on the clock genes in LO2 cells and its significance. Methods： A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Resuits： The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx （P 〈 0.05）, while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx （P 〈 0.05）. Conclusion： The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer.

Prevalence of hepatocarcinoma-related hepatitis B virus mutants in patients in grey zone of treatment

BACKGROUND Antiviral treatment of patients with chronic hepatitis B(CHB)in the grey zone of treatment comands risk management in order to optimize the health outcome.In this sense,the identification of HBV mutants related with an increased risk of hepatocellular carcinoma(HCC)could be useful to identify subpopulations with potential indication of antiviral treatment.AIM To analyze the prevalence/persistence of hepatitis B virus(HBV)preS and basal core promoter(BCP)/precore/core variants associated to HCC development in CHB patients in the grey zone.METHODS Work was designed as a longitudinal retrospective study,including 106 plasma samples from 31 patients with CHB in the grey zone of treatment:Hepatitis B e antigen negative,HBV-DNA levels between 12-20000 IU/mL,normal or discordant transaminase levels during follow up and mild/moderate necroinflammatory activity in liver biopsy or Fibroscan(up to 9.5 kPa).Serum HBVDNA was tested using the Abbott Real Time HBV Assay and the BCP/precore/core and the hepatitis B surface antigen(HBsAg)coding regions were analyzed in positive samples by PCR/bulk-sequencing to identify the HCCrelated HBV mutants.RESULTS High-risk HCC related mutants were detected in 24(77%)patients:19(61%)in the BCP/precore/core,and 7(23%)in the HBsAg coding region(2 preS1 and 5 preS2 deletions).The prevalence of preS deletions was genotype-dependent:3/5(60%)patients with preS2 deletions and 1/2 with preS1 deletions were infected with the HBV-E genotype.Since HBV-E was the most prevalent in sub-Saharan patients,a correlation between preS deletions and ethnicity was also found:6/8(75%)sub-Saharan vs 1/19(5%)Caucasian patients had preS deletions(P=0.00016).Remarkably,this correlation was maintained in those patients infected with HBV-A,a minor genotype in sub-Saharan patients:2/2 patients infected with HBV-A from West Africa vs 0/6 of Caucasian origin had preS deletions.The HCC related variants were the major strains and persisted over time(up to 48 mo).Patients with preS deletions had a significant higher prevalence of F2 fibrosis stage than the negatives(57%vs 10%,P=0.0078).CONCLUSION HBV genetic analysis of selected populations,like sub-Saharans infected with HBV-E/A genotypes,will allow identification of subpopulations with risk of HCC development due to accumulation of high-risk HBV variants,thus commanding their increased clinical surveillance.

Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma

AIM: To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers.