To evaluate the effects of duck hepatitis virus-1 (DHV-1) on the body weight gain in duck and the effects of silymarin on it in vivo, 100 10-d-old ducks, both male and female, were collected to be subjected to the t...To evaluate the effects of duck hepatitis virus-1 (DHV-1) on the body weight gain in duck and the effects of silymarin on it in vivo, 100 10-d-old ducks, both male and female, were collected to be subjected to the test. The experiments were conducted in 8 groups: in group 1-3, the animals were inoculated with 1:105 diluted duck hepatitis virus (DHV-1) infected allantoic fluid and given 0, 30, and 50 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 4-6, the animals were inoculated with 1:5 × 105 diluted DHV-1 infected allantoic fluid and given 0, 10, and 30 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 7, the animals were given 10 mg kg^-1 BW d^-1 silymafin only. Group 8 was the control one treated by injecting sterillized saline into the leg muscles. All the silymarin was given from 0 to 4 d after inoculation of the virus. By the 5th d after inoculation, the vein blood was drawn from the dorsal foot vein and the plasma samples were collected and stored at -20℃. The body weight gain (BWG) was measured from 0 to 10 d after inoculation. The plasma IGF-I, T3, and T4 concentrations were measured by radioimmunoassay (RIA). At the virus dose of 1:5 ×105 diluted virus infected allantoic fluid, the inoculation of the virus enhanced the BWG significantly compared with that of the control (P〈 0.01), while 10-50 mg kg^-1 BW d^-1 silymarin could counteract the effects of the virus on the BWG dose-dependently. The plasma IGF-I levels showed no correlation with the BWG, but the T3 levels showed a same tropism with the body weight gain. The present results indicated that sublethal DHV-1 enhanced the body weight gain of ducklings significantly, and the silymarin could counteract this effect in vivo.展开更多
To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete ...To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.展开更多
To determine the genomic sequence of a duck hepatitis virus type 1 (DHV-1) strain, real-time quantitative polymerase chain reaction (RTQ-PCR) assay based on SYBR Green I technology was developed to target 3D gene ...To determine the genomic sequence of a duck hepatitis virus type 1 (DHV-1) strain, real-time quantitative polymerase chain reaction (RTQ-PCR) assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5' untranslated region (UTR)-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.展开更多
In this study,a RT-PCR assay for rapid detection of duck hepatitis virus type 1( DHV-1) was established in the presence of a pair of primers designed based on vp1 gene sequence,using the genomic RNA of DHV-1 or other ...In this study,a RT-PCR assay for rapid detection of duck hepatitis virus type 1( DHV-1) was established in the presence of a pair of primers designed based on vp1 gene sequence,using the genomic RNA of DHV-1 or other common viruses as the template. The template was serially diluted 10-fold to determine the reaction sensitivity. Finally,seven liver samples of ducks with suspected DHV-1 infection,which were collected from different regions of Jiangsu Province,were detected with this method. The results showed that a DNA fragment of about 360 bp was specifically amplified with the RT-PCR system,and the detection limit was1 fg/μL. The detection rate of clinical samples with this method was 100%. The results revealed that the RT-PCR system which had high specificity and high sensitivity in the detection of DHV-1 was successfully established.展开更多
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 14...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.展开更多
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 ch...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.展开更多
Chronic hepatitis C virus(HCV) infection is a public health issue that often progresses to life-threatening complications, including liver cirrhosis, fibrosis, and hepatocellular carcinoma. Impaired immune responses t...Chronic hepatitis C virus(HCV) infection is a public health issue that often progresses to life-threatening complications, including liver cirrhosis, fibrosis, and hepatocellular carcinoma. Impaired immune responses to HCV are key features of chronic HCV infection. Therefore, intervention strategies usually involve enhancing the immune responses against HCV. Cytotoxic CD8+ T lymphocytes(CTLs) play a critical role in the control of HCV infection. However, their cytolytic function can be impaired by the expression of co-inhibitory molecules. Programmed death-1(PD-1) receptor and its ligand PD-L1 function in a T cell co-inhibitory pathway, which either blocks the function of CTLs or the differentiation of CD8+ T cells. During chronic HCV infection, the immune inhibitory receptor PD-1 is upregulated on dysfunctional HCV-specific CD8+ T cells. As such, blockade of the PD-1/PD-L1 pathway in these CD8+ T cells might restore their functional capabilities. Indeed, clinical trials using therapies to block this pathway have shown promise in the fostering of anti-HCV immunity. Understanding how chronic HCV infection induces upregulation of PD-1 on HCV specific T cells and how the PD-1/PD-L1 interaction develops HCV specific T cell dysfunction will accelerate the development of an efficacious prophylactic and therapeutic vaccination against chronic HCV infections, which will significantly improve HCV treatments and patient survival. In this review, we discuss the relationship between PD-1 expression and clinical responses and the potential use of PD-1 blockade for anti-HCV therapy.展开更多
Hepatitis C virus(HCV) infection and diabetes mellitus are two major public health problems that cause devastating health and financial burdens worldwide. Diabetes can be classified into two major types: type 1 diabet...Hepatitis C virus(HCV) infection and diabetes mellitus are two major public health problems that cause devastating health and financial burdens worldwide. Diabetes can be classified into two major types: type 1 diabetes mellitus(T1DM) and T2 DM. T2 DM is a common endocrine disorder that encompasses multifactorial mechanisms, and T1 DM is an immunologically mediated disease. Many epidemiological studies have shown an association between T2 DM and chronic hepatitis C(CHC) infection. The processes through which CHC is associated with T2 DM seem to involve direct viral effects, insulin resistance, proinflammatory cytokines, chemokines, and other immunemediated mechanisms. Few data have been reported on the association of CHC and T1 DM and reports on the potential association between T1 DM and acute HCV infection are even rarer. A small number of studies indicate that interferon-α therapy can stimulate pancreatic autoim-munity and in certain cases lead to the development of T1 DM. Diabetes and CHC have important interactions. Diabetic CHC patients have an increased risk of developing cirrhosis and hepatocellular carcinoma compared with nondiabetic CHC subjects. However, clinical trials on HCV-positive patients have reported improvements in glucose metabolism after antiviral treatment. Further studies are needed to improve prevention policies and to foster adequate and cost-effec-tive programmes for the surveillance and treatment of diabetic CHC patients.展开更多
The mechanism of action of ribavirin(RBV) as an immunomodulatory and antiviral agent and its clinical significance in the future treatment of patients with hepatitis C virus(HCV) infection are reviewed.RBV up-regulate...The mechanism of action of ribavirin(RBV) as an immunomodulatory and antiviral agent and its clinical significance in the future treatment of patients with hepatitis C virus(HCV) infection are reviewed.RBV up-regulates type 1 and/or 2 cytokines to modulate the T helper(Th) 1/2 cell balance to Th1 dominance.Examination of co-stimulatory signaling indicated that RBV down-modulates inducible co-stimulator on Th cells,which contributes to differentiating na?ve Th cells into Th2 cells while reducing their interleukin-10 production.The effects on T-regulatory(Treg) cells were also investigated,and RBV inhibited the differentiation of na?ve Th cells into adaptive Treg cells by downmodulating forkhead box-P3.These findings indicate that RBV mainly down-regulates the activity of Th2 cells,resulting in the maintenance of Th1 activity that contributes to abrogating HCV-infected hepatocytes.Although an interferon-free treatment regimen exhibits almost the same efficacy without serious complications,regimens with RBV will be still be used because of their ability to facilitate the cellular immune response,which may contribute to reducing the development of hepatocellular carcinogenesis in patients infected with HCV.展开更多
AIM:To investigate the clinical implications of hepatitis B virus(HBV) pre S1 deletion.METHODS:We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction(RT-PCR) that can detect fo...AIM:To investigate the clinical implications of hepatitis B virus(HBV) pre S1 deletion.METHODS:We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction(RT-PCR) that can detect four genotypes(wild type, 15-bp, 18-bp and 21-bp deletion).The PCR method was used in two cohorts of Korean chronic HBV subjects with genotype C infections.Cohort Ⅰ included 292 chronic HBV subjects randomly selected from Cheju National University Hospital(Jeju, South Korea) or Seoul National University Hospital(Seoul, South Korea), and cohort Ⅱ included 90 consecutive chronic HBV carriers recruited from Konkuk University Hospital(Seoul, South Korea); the cohort Ⅱ patients did not have hepatocellular carcinoma or liver cirrhosis.RESULTS:The method proposed in this study identified 341 of 382 samples(89.3%).Deletion variants were identified in 100(29.3%) of the 341 detected samples.In both cohorts, the subjects with deletions had a significantly higher Hepatitis B virus e antigen(HBe Ag)-positive seroprevalence [cohort Ⅰ, wild(51.0%) vs deletion(75.0%), P < 0.001; cohort Ⅱ, wild(69.2%) vs deletion(92.9%), P = 0.002] and higher HBV DNA levels [cohort Ⅰ, wild(797.7 pg/m L) vs deletion(1678.9 pg/m L), P = 0.013; cohort Ⅱ, wild(8.3 × 108 copies/m L) vs deletion(2.2 × 109 copies/m L), P = 0.049], compared to subjects with wild type HBV.CONCLUSION:HBV genotype C pre S1 deletion may affect disease progression in chronic HBV subjects through an extended duration of HBe Ag seropositive status and increased HBV replications.展开更多
Objective: The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting of NUSAP1 by miR-18b to enhance hepatocarcinogenesis.Methods: We employed an integrated approach of bioi...Objective: The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting of NUSAP1 by miR-18b to enhance hepatocarcinogenesis.Methods: We employed an integrated approach of bioinformatics analysis and molecular experiments in hepatoma cells, HBV transgenic mice, and clinical liver cancer tissues to investigate the role of HBx-regulated miR-18b in the development of liver cancer.Results: In this study, we report that the HBx-mediated tumor suppressor miR-18b modulates hepatocarcinogenesis during the host-HBV interaction. The expression levels of miR-18b were lower in clinical HBV-positive liver cancer tissues and liver tissues of HBV-transgenic mice. Interestingly, HBx inhibited miR-18b expression by inducing the methylation of CpG islands in its promoter. Accordingly, we tested the hypothesis that HBx enhanced hepatocarcinogenesis by increasing the expression of target genes of miR-18b. Moreover, we identified nucleolar spindle-associated protein 1(NUSAP1) as one of the target genes of miR-18b.NUSAP1 was expressed at high levels in liver cancer tissues. Interestingly, HBx up-regulated NUSAP1 by suppressing miR-18b.Functionally, miR-18b significantly inhibited the proliferation of hepatoma cells by depressing NUSAP1 levels in vivo and in vitro.Conclusions: Thus, we conclude that the targeting of NUSAP1 mRNA by the tumor suppressor miR-18b is controlled by HBxmodulated promoter methylation during the host-virus interaction, leading to hepatocarcinogenesis. Our findings provide new insights into the mechanism by which HBx-mediated miRNAs modulate hepatocarcinogenesis.展开更多
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 we...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.展开更多
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 preval...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.展开更多
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...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.展开更多
AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in...AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.展开更多
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 p...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.展开更多
BACKGROUND The expression of macrophage inhibitory factor-1(MIC-1) is increased in peripheral blood of patients with chronic hepatitis and liver cirrhosis. However, whether MIC-1 gene polymorphism is correlated with r...BACKGROUND The expression of macrophage inhibitory factor-1(MIC-1) is increased in peripheral blood of patients with chronic hepatitis and liver cirrhosis. However, whether MIC-1 gene polymorphism is correlated with relevant diseases is not yet reported.AIM To explore the correlation between gene polymorphism in MIC-1 exon region and chronic hepatitis C virus(HCV) infection.METHODS This case-control study enrolled 178 patients with chronic hepatitis C(CHC) in the case group, and 82 healthy subjects from the same region who had passed the screening examination comprised the control group. The genotypes of rs1059369 and rs1059519 loci in the MIC-1 gene exon were detected by DNA sequencing. Also, the MIC-1 level, liver function metrics, liver fibrosis metrics, and HCV RNA load were determined. Univariate analysis was used to compare the differences and correlations between the two groups with respect to these parameters. Multivariate logistic regression was used to analyze the independent relevant factors of CHC.RESULTS The plasma MIC-1 level in the CHC group was higher than that in the control group(P < 0.05), and it was significantly positively correlated with alanine aminotransferase, aspartate aminotransferase(AST), type III procollagen N-terminal peptide(known as PIIINP), type IV collagen, and HCV RNA(P < 0.05), whereas negatively correlated with total protein and albumin(P < 0.05). The genotype and allele frequency distribution at the rs1059519 locus differed between the two groups(P < 0.05). The allele frequency maintained significant difference after Bonferroni correction(Pc < 0.05). Logistic multiple regression showed that AST, PIIINP, MIC-1, and genotype GG at the rs1059519 locus were independent relevant factors of CHC(P < 0.05). Linkage disequilibrium(LD) was found between rs1059369 and rs1059519 loci, and significant difference was detected in the distribution of haplotype A-C between the CHC and control groups(P < 0.05). Meanwhile, we found the MIC-1 level trend to increase among rs1059519 genotypes(P = 0.006) and the level of MIC-1 in GG genotype to be significantly higher than CC genotype(P = 0.009, after Bonferroni correction).CONCLUSION Plasma MIC-1 level was increased in CHC patients and correlated with liver cell damage, liver fibrosis metrics, and viral load. The polymorphism at the MIC-1 gene rs1059519 locus was correlated with HCV infection, and associated with the plasma MIC-1 level. G allele and GG genotype may be an important susceptible factor for CHC.展开更多
AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine(SAM) decreases hepatitis C virus(HCV) expression.METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell cu...AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine(SAM) decreases hepatitis C virus(HCV) expression.METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times(24-72 h), then total RNA and proteins were isolated. c DNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2(SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A(MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5 A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1 A, MAT2 A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method(0-24 h). Reactive oxidative species(ROS) levels were quantified by the dichlorofluorescein assay(0-48 h); Pyrrolidin dithiocarbamate(PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent.RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls(24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression(SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2 A, since MAT1 A expression was increased(2.5 fold-times at 48 h) and MAT2 A was diminished(from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.展开更多
AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell c...AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.展开更多
基金supported by the National Natural Science Foundation of China (30571341)Foshan Science and Technology Development Programme,China(04040111)
文摘To evaluate the effects of duck hepatitis virus-1 (DHV-1) on the body weight gain in duck and the effects of silymarin on it in vivo, 100 10-d-old ducks, both male and female, were collected to be subjected to the test. The experiments were conducted in 8 groups: in group 1-3, the animals were inoculated with 1:105 diluted duck hepatitis virus (DHV-1) infected allantoic fluid and given 0, 30, and 50 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 4-6, the animals were inoculated with 1:5 × 105 diluted DHV-1 infected allantoic fluid and given 0, 10, and 30 mg kg^-1 BW d^-1 silymarin orally, respectively. In group 7, the animals were given 10 mg kg^-1 BW d^-1 silymafin only. Group 8 was the control one treated by injecting sterillized saline into the leg muscles. All the silymarin was given from 0 to 4 d after inoculation of the virus. By the 5th d after inoculation, the vein blood was drawn from the dorsal foot vein and the plasma samples were collected and stored at -20℃. The body weight gain (BWG) was measured from 0 to 10 d after inoculation. The plasma IGF-I, T3, and T4 concentrations were measured by radioimmunoassay (RIA). At the virus dose of 1:5 ×105 diluted virus infected allantoic fluid, the inoculation of the virus enhanced the BWG significantly compared with that of the control (P〈 0.01), while 10-50 mg kg^-1 BW d^-1 silymarin could counteract the effects of the virus on the BWG dose-dependently. The plasma IGF-I levels showed no correlation with the BWG, but the T3 levels showed a same tropism with the body weight gain. The present results indicated that sublethal DHV-1 enhanced the body weight gain of ducklings significantly, and the silymarin could counteract this effect in vivo.
基金the Chinese National Natural Sciences Foundation(30871878)Shandong Province Higher Educational Science and Technology Program(J08LF07)the Science and Technology Commission of Shandong Province(2010GNC10914),China
文摘To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.
基金the Natural Science Foundation of Guangdong Province,China (5006678)the National Natural Science Foundation of China
文摘To determine the genomic sequence of a duck hepatitis virus type 1 (DHV-1) strain, real-time quantitative polymerase chain reaction (RTQ-PCR) assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5' untranslated region (UTR)-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.
基金Supported by Natural Science Research Program of the Higher Education Institutions of Jiangsu Province(16KJB230004)"Six Talent Peaks" Project of Jiangsu Province(NY-023,10410015002)+1 种基金Anhui Science and Technology Project(1201c0602006)Jiangsu Province Excellent Scientific and Technological Innovation Tean
文摘In this study,a RT-PCR assay for rapid detection of duck hepatitis virus type 1( DHV-1) was established in the presence of a pair of primers designed based on vp1 gene sequence,using the genomic RNA of DHV-1 or other common viruses as the template. The template was serially diluted 10-fold to determine the reaction sensitivity. Finally,seven liver samples of ducks with suspected DHV-1 infection,which were collected from different regions of Jiangsu Province,were detected with this method. The results showed that a DNA fragment of about 360 bp was specifically amplified with the RT-PCR system,and the detection limit was1 fg/μL. The detection rate of clinical samples with this method was 100%. The results revealed that the RT-PCR system which had high specificity and high sensitivity in the detection of DHV-1 was successfully established.
基金Supported by The National Natural Science Foundation of China,grant No.30972598The State Key Project Specialized for Infectious Diseases,No.2012ZX10002-004the TMMU Key Project for Clinical Study,No.2012XLC-005
文摘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.
文摘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.
基金Supported by Science and Technology Development Fund(STDFgrants No.1469 and No.5245)Tanta University Fund,Egypt to Mohamed L Salem,the Principal investigator of these projects
文摘Chronic hepatitis C virus(HCV) infection is a public health issue that often progresses to life-threatening complications, including liver cirrhosis, fibrosis, and hepatocellular carcinoma. Impaired immune responses to HCV are key features of chronic HCV infection. Therefore, intervention strategies usually involve enhancing the immune responses against HCV. Cytotoxic CD8+ T lymphocytes(CTLs) play a critical role in the control of HCV infection. However, their cytolytic function can be impaired by the expression of co-inhibitory molecules. Programmed death-1(PD-1) receptor and its ligand PD-L1 function in a T cell co-inhibitory pathway, which either blocks the function of CTLs or the differentiation of CD8+ T cells. During chronic HCV infection, the immune inhibitory receptor PD-1 is upregulated on dysfunctional HCV-specific CD8+ T cells. As such, blockade of the PD-1/PD-L1 pathway in these CD8+ T cells might restore their functional capabilities. Indeed, clinical trials using therapies to block this pathway have shown promise in the fostering of anti-HCV immunity. Understanding how chronic HCV infection induces upregulation of PD-1 on HCV specific T cells and how the PD-1/PD-L1 interaction develops HCV specific T cell dysfunction will accelerate the development of an efficacious prophylactic and therapeutic vaccination against chronic HCV infections, which will significantly improve HCV treatments and patient survival. In this review, we discuss the relationship between PD-1 expression and clinical responses and the potential use of PD-1 blockade for anti-HCV therapy.
文摘Hepatitis C virus(HCV) infection and diabetes mellitus are two major public health problems that cause devastating health and financial burdens worldwide. Diabetes can be classified into two major types: type 1 diabetes mellitus(T1DM) and T2 DM. T2 DM is a common endocrine disorder that encompasses multifactorial mechanisms, and T1 DM is an immunologically mediated disease. Many epidemiological studies have shown an association between T2 DM and chronic hepatitis C(CHC) infection. The processes through which CHC is associated with T2 DM seem to involve direct viral effects, insulin resistance, proinflammatory cytokines, chemokines, and other immunemediated mechanisms. Few data have been reported on the association of CHC and T1 DM and reports on the potential association between T1 DM and acute HCV infection are even rarer. A small number of studies indicate that interferon-α therapy can stimulate pancreatic autoim-munity and in certain cases lead to the development of T1 DM. Diabetes and CHC have important interactions. Diabetic CHC patients have an increased risk of developing cirrhosis and hepatocellular carcinoma compared with nondiabetic CHC subjects. However, clinical trials on HCV-positive patients have reported improvements in glucose metabolism after antiviral treatment. Further studies are needed to improve prevention policies and to foster adequate and cost-effec-tive programmes for the surveillance and treatment of diabetic CHC patients.
文摘The mechanism of action of ribavirin(RBV) as an immunomodulatory and antiviral agent and its clinical significance in the future treatment of patients with hepatitis C virus(HCV) infection are reviewed.RBV up-regulates type 1 and/or 2 cytokines to modulate the T helper(Th) 1/2 cell balance to Th1 dominance.Examination of co-stimulatory signaling indicated that RBV down-modulates inducible co-stimulator on Th cells,which contributes to differentiating na?ve Th cells into Th2 cells while reducing their interleukin-10 production.The effects on T-regulatory(Treg) cells were also investigated,and RBV inhibited the differentiation of na?ve Th cells into adaptive Treg cells by downmodulating forkhead box-P3.These findings indicate that RBV mainly down-regulates the activity of Th2 cells,resulting in the maintenance of Th1 activity that contributes to abrogating HCV-infected hepatocytes.Although an interferon-free treatment regimen exhibits almost the same efficacy without serious complications,regimens with RBV will be still be used because of their ability to facilitate the cellular immune response,which may contribute to reducing the development of hepatocellular carcinogenesis in patients infected with HCV.
基金Supported by Grants from National Research Foundation of Koreagrant funded by the Korean government(Ministry of Education,Science,and Technology),No.2013-005810Foundation of Seoul National University Hospital(SNUH research fund),No.0320140140
文摘AIM:To investigate the clinical implications of hepatitis B virus(HBV) pre S1 deletion.METHODS:We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction(RT-PCR) that can detect four genotypes(wild type, 15-bp, 18-bp and 21-bp deletion).The PCR method was used in two cohorts of Korean chronic HBV subjects with genotype C infections.Cohort Ⅰ included 292 chronic HBV subjects randomly selected from Cheju National University Hospital(Jeju, South Korea) or Seoul National University Hospital(Seoul, South Korea), and cohort Ⅱ included 90 consecutive chronic HBV carriers recruited from Konkuk University Hospital(Seoul, South Korea); the cohort Ⅱ patients did not have hepatocellular carcinoma or liver cirrhosis.RESULTS:The method proposed in this study identified 341 of 382 samples(89.3%).Deletion variants were identified in 100(29.3%) of the 341 detected samples.In both cohorts, the subjects with deletions had a significantly higher Hepatitis B virus e antigen(HBe Ag)-positive seroprevalence [cohort Ⅰ, wild(51.0%) vs deletion(75.0%), P < 0.001; cohort Ⅱ, wild(69.2%) vs deletion(92.9%), P = 0.002] and higher HBV DNA levels [cohort Ⅰ, wild(797.7 pg/m L) vs deletion(1678.9 pg/m L), P = 0.013; cohort Ⅱ, wild(8.3 × 108 copies/m L) vs deletion(2.2 × 109 copies/m L), P = 0.049], compared to subjects with wild type HBV.CONCLUSION:HBV genotype C pre S1 deletion may affect disease progression in chronic HBV subjects through an extended duration of HBe Ag seropositive status and increased HBV replications.
基金supported in part by grants from National Basic Research Program of China (973 Program, Grant No. 2015CB553703)National Natural Science Foundation of China (Grant No. 31670769 and 31470756)
文摘Objective: The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting of NUSAP1 by miR-18b to enhance hepatocarcinogenesis.Methods: We employed an integrated approach of bioinformatics analysis and molecular experiments in hepatoma cells, HBV transgenic mice, and clinical liver cancer tissues to investigate the role of HBx-regulated miR-18b in the development of liver cancer.Results: In this study, we report that the HBx-mediated tumor suppressor miR-18b modulates hepatocarcinogenesis during the host-HBV interaction. The expression levels of miR-18b were lower in clinical HBV-positive liver cancer tissues and liver tissues of HBV-transgenic mice. Interestingly, HBx inhibited miR-18b expression by inducing the methylation of CpG islands in its promoter. Accordingly, we tested the hypothesis that HBx enhanced hepatocarcinogenesis by increasing the expression of target genes of miR-18b. Moreover, we identified nucleolar spindle-associated protein 1(NUSAP1) as one of the target genes of miR-18b.NUSAP1 was expressed at high levels in liver cancer tissues. Interestingly, HBx up-regulated NUSAP1 by suppressing miR-18b.Functionally, miR-18b significantly inhibited the proliferation of hepatoma cells by depressing NUSAP1 levels in vivo and in vitro.Conclusions: Thus, we conclude that the targeting of NUSAP1 mRNA by the tumor suppressor miR-18b is controlled by HBxmodulated promoter methylation during the host-virus interaction, leading to hepatocarcinogenesis. Our findings provide new insights into the mechanism by which HBx-mediated miRNAs modulate hepatocarcinogenesis.
基金Supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute and the Ministry of Health and Welfare,South Korea,No.HI14C0955
文摘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.
基金Supported by Instituto de Salud Carlos Ⅲ,No.PI14/01416 and No.PI15/00856cofinanced by the European Regional Development Fund(ERDF)the Gilead Fellowship Program,No.GLD14-00296
文摘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.
文摘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.
基金Supported by National Natural Science Foundation of China,No.81371849the TMMU Key Project for Clinical Research,No.2012XLC05
文摘AIM To investigate the mechanism by which hepatitis C virus(HCV) core protein-induced mi R-93-5 p up-regulation regulates the interferon(IFN) signaling pathway.METHODS HCV-1 b core protein was exogenously expressed in Huh7 cells using pc DNA3.1(+) vector. The expression of mi R-93-5 p and interferon receptor 1(IFNAR1) was measured using quantitative reverse transcriptionpolymerase chain reaction and Western blot. The protein expression and phosphorylation level of STAT1 were evaluated by Western blot. The overexpression and silencing of mi R-93-5 p and IFNAR1 were performed using mi R-93-5 p agomir and antagomir, and pc DNA3.1-IFNAR1 and IFNAR1 si RNA, respectively. Luciferase assay was used to identify whether IFNAR1 is a target of mi R-93-5 p. Cellular experiments were also conducted.RESULTS Serum mi R-93-5 p level was increased in patients with HCV-1 b infection and decreased to normal level after HCV-1 b clearance, but persistently increased in those with pegylated interferon-α resistance, compared with healthy subjects. Serum mi R-93-5 p expression had an AUC value of 0.8359 in distinguishing patients with pegylated interferon-α resistance from those with pegylated interferon-α sensitivity. HCV-1 b core protein increased mi R-93-5 p expression and induced inactivation of the IFN signaling pathway in Huh7 cells. Furthermore, IFNAR1 was identified as a direct target of mi R-93-5 p, and IFNAR1 restore could rescue mi R-93-5 p-reduced STAT1 phosphorylation, suggesting that the mi R-93-5 p-IFNAR1 axis regulates the IFN signaling pathway.CONCLUSION HCV-1 b core protein-induced mi R-93-5 p up-regulation inhibits the IFN signaling pathway by directly targeting IFNAR1, and the mi R-93-5 p-IFNAR1 axis regulates STAT1 phosphorylation. This axis may be a potential therapeutic target for HCV-1 b infection.
文摘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.
基金Supported by the Medical and Health Research Science and Technology Plan Project of Zhejiang Province,No. 2016KYB191。
文摘BACKGROUND The expression of macrophage inhibitory factor-1(MIC-1) is increased in peripheral blood of patients with chronic hepatitis and liver cirrhosis. However, whether MIC-1 gene polymorphism is correlated with relevant diseases is not yet reported.AIM To explore the correlation between gene polymorphism in MIC-1 exon region and chronic hepatitis C virus(HCV) infection.METHODS This case-control study enrolled 178 patients with chronic hepatitis C(CHC) in the case group, and 82 healthy subjects from the same region who had passed the screening examination comprised the control group. The genotypes of rs1059369 and rs1059519 loci in the MIC-1 gene exon were detected by DNA sequencing. Also, the MIC-1 level, liver function metrics, liver fibrosis metrics, and HCV RNA load were determined. Univariate analysis was used to compare the differences and correlations between the two groups with respect to these parameters. Multivariate logistic regression was used to analyze the independent relevant factors of CHC.RESULTS The plasma MIC-1 level in the CHC group was higher than that in the control group(P < 0.05), and it was significantly positively correlated with alanine aminotransferase, aspartate aminotransferase(AST), type III procollagen N-terminal peptide(known as PIIINP), type IV collagen, and HCV RNA(P < 0.05), whereas negatively correlated with total protein and albumin(P < 0.05). The genotype and allele frequency distribution at the rs1059519 locus differed between the two groups(P < 0.05). The allele frequency maintained significant difference after Bonferroni correction(Pc < 0.05). Logistic multiple regression showed that AST, PIIINP, MIC-1, and genotype GG at the rs1059519 locus were independent relevant factors of CHC(P < 0.05). Linkage disequilibrium(LD) was found between rs1059369 and rs1059519 loci, and significant difference was detected in the distribution of haplotype A-C between the CHC and control groups(P < 0.05). Meanwhile, we found the MIC-1 level trend to increase among rs1059519 genotypes(P = 0.006) and the level of MIC-1 in GG genotype to be significantly higher than CC genotype(P = 0.009, after Bonferroni correction).CONCLUSION Plasma MIC-1 level was increased in CHC patients and correlated with liver cell damage, liver fibrosis metrics, and viral load. The polymorphism at the MIC-1 gene rs1059519 locus was correlated with HCV infection, and associated with the plasma MIC-1 level. G allele and GG genotype may be an important susceptible factor for CHC.
基金Supported by CONACYT-Mexico,grant register CB2010-01-155082 to Rivas-Estilla AM
文摘AIM: To elucidate the mechanism(s) by which S-adenosyl-L-methionine(SAM) decreases hepatitis C virus(HCV) expression.METHODS: We examined the effects of SAM on viral expression using an HCV subgenomic replicon cell culture system. Huh7 HCV-replicon cells were treated with 1 mmol/L SAM for different times(24-72 h), then total RNA and proteins were isolated. c DNA was synthesized and real time-PCR was achieved to quantify HCV-RNA, superoxide dismutase 1 and 2(SOD-1, SOD-2) catalase, thioredoxin 1, methionine adenosyltransferase 1A and 2A(MAT1A, MAT2A) expression, and GAPDH and RPS18 as endogenous genes. Expression of cellular and viral protein was evaluated by western-blot analysis using antibodies vs HCV-NS5 A, SOD-1, SOD-2, catalase, thioredoxin-1, MAT1 A, MAT2 A, GAPDH and actin. Total glutathione levels were measured at different times by Ellman's recycling method(0-24 h). Reactive oxidative species(ROS) levels were quantified by the dichlorofluorescein assay(0-48 h); Pyrrolidin dithiocarbamate(PDTC) was tested as an antioxidant control and H2O2 as a positive oxidant agent.RESULTS: SAM exposition decreased HCV-RNA levels 50%-70% compared to non-treated controls(24-72 h). SAM induced a synergic antiviral effect with standard IFN treatment but it was independent of IFN signaling. In addition, 1 mmol/L SAM exposition did not modify viral RNA stability, but it needs cellular translation machinery in order to decrease HCV expression. Total glutathione levels increased upon SAM treatment in HCV-replicon cells. Transcriptional antioxidant enzyme expression(SOD-1, SOD-2 and thioredoxin-1) was increased at different times but interestingly, there was no significant change in ROS levels upon SAM treatment, contrary to what was detected with PDTC treatment, where an average 40% reduction was observed in exposed cells. There was a turnover from MAT1A/MAT2 A, since MAT1 A expression was increased(2.5 fold-times at 48 h) and MAT2 A was diminished(from 24 h) upon SAM treatment at both the transcriptional and translational level. CONCLUSION: A likely mechanism(s) by which SAM diminish HCV expression could involve modulating antioxidant enzymes, restoring biosynthesis of glutathione and switching MAT1/MAT2 turnover in HCV expressing cells.
基金Supported by the National Natural Science Foundation of China,No.81500449the Natural Science Foundation of Shanghai,No.14ZR1434200+2 种基金Shanghai Municipal Commission of Health and Family Planning,No.20144Y0175the Scientific Research Foundation for the Returned Overseas Chinese Scholarsthe State Education Ministry of China,No.20150909-6
文摘AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.