AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraper...AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinaseactivity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice.In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2)was detected in 21 of 23 patients (91.30%)with severe AOC hepatitis B, while only 1 of 13 patients(7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1of 10 with mild chronic hepatitis B had detectable hfgl2expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2protein play a pivotal role in initiating severe hepatitis.The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.展开更多
Hepatitis C virus(HCV)infection is the leading cause of chronic liver-related diseases,including cirrhosis,liver failure,and hepatocellular carcinoma.Currently,no effective vaccine is available for HCV infection.Polye...Hepatitis C virus(HCV)infection is the leading cause of chronic liver-related diseases,including cirrhosis,liver failure,and hepatocellular carcinoma.Currently,no effective vaccine is available for HCV infection.Polyethylene glycol interferon-α(PegIFN-α)in combination with ribavirin(RBV)is the standard of care(SOC)for chronic hepatitis C.However,the efficacy of PegIFN-αand RBV combination therapy is less than 50%for genotype 1HCV,which is the dominant virus in humans.In addition,IFN and RBV have several severe side effects.Therefore,strategies to improve sustained virological response(SVR)rates have been an important focus for clinical physicians.The serine protease inhibitors telaprevir and boceprevir were approved by the United States Food and Drug Administration in 2011.The addition of HCV protease inhibitors to the SOC has significantly improved the efficacy of treatments for HCV infection.Several direct-acting antiviral drugs currently in late-stage clinical trials,both with and without pegIFN and RBV,have several advantages over the previous SOC,including higher specificity and efficacy,fewer side effects,and the ability to be administered orally,and might be optimal regimens in the future.Factors affecting the efficacy of anti-HCV treatments based on IFN-αinclude the HCV genotype,baseline viral load,virological response during treatment,host IL28B gene polymorphisms and hepatic steatosis.However,determining the effect of the above factors on DAA therapy is necessary.In this review,we summarize the development of antiHCV agents and assess the main factors affecting the efficacy of antiviral treatments.展开更多
AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragmen...AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.展开更多
The mechanisms involved in virus-induced severe hepatitis have not been fully elucidated. In this study, we investigated the role of gamma delta T cell receptors (γδ) T cells in the pathogenesis of fulminant viral...The mechanisms involved in virus-induced severe hepatitis have not been fully elucidated. In this study, we investigated the role of gamma delta T cell receptors (γδ) T cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). The model of FVH was established by intraperitoneal injection of MHV-3 into Balb/cJ mice. The survival days of mice, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined. The proportions of γδ T cells in blood, spleen and liver, and cytokines secreted by hepatic γδ T cells were analyzed by flow cytometry. The function of hepatic γδ T cells was examined by cytotoxicity assay. Balb/cJ mice died in 3 to 6 days post MHV-3 infection, with severe hepatic necrosis and significant augmentation of serum ALT and AST levels. The proportions of γδ T cells in blood, spleen and liver were significantly increased post MHV-3 infection, while those of the early activating molecule CD69-expressing γδ T cells and productions of cytokines tumor necrosis factor-alpha (TNF-α) and interferon-y (IFN-3,) increased remarkably in the liver. These highly activated liver γδ T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect of liver γδ T cells against hepatocytes might involve the TNF-α and IFN-γ pathway. These results demonstrated that γδ T cells might contribute to the pathogenesis of MHV-3-induced FVH through the effector cytokines TNF-α and IFN-γ. Key words: fulminant viral hepatitis; murine hepatitis virus strain 3; gamma delta T cell receptors T cells; tumor necrosis factor-a; interferon-α展开更多
AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected wi...AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected with HCV or transientlytransfected with a vector expressing HCV NS5 A. The effect of HCV NS5 A on the status of the critical players involved in insulin signaling was analyzed using realtime quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0.RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway,we analyzed the status of insulin receptor substrate-1(IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5 A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1(Ser307) in HCV infected or NS5 A transfected Huh7.5 cells compared to their respective controls. Furthermore,an increased phosphorylation of Akt(Ser473) was observed in HCV infected and NS5 A transfected cells compared to their mock infected cells. In contrast,we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5 A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5 A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue(Ser307) followed by decreased phosphorylation of Akt Thr308,Fox01 Ser256 and GSK3β Ser9,the downstream players of the insulin signalingpathway. Furthermore,increased expression of PECK and glucose-6-phosphatase,the molecules involved in gluconeogenesis,in HCV NS5 A transfected cells was observed.CONCLUSION: Taken together,our results suggest the role of HCV NS5 A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.展开更多
The hepatitis C virus(HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases includ...The hepatitis C virus(HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases:(1) binding and internalization;(2) cytoplasmic release and uncoating;(3) viral polyprotein translation and processing;(4) RNA genome replication;(5) encapsidation(packaging) and assembly; and(6) virus morphogenesis(maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.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 study persistence and replication ofheltitis C virus (HCV) in patients' peripheralblood mononuclear cells (PBMC) cultured invitro.METHODS Epstein-Barr virus (EBV) was usedto transform the hepatitis C virus ...AIM To study persistence and replication ofheltitis C virus (HCV) in patients' peripheralblood mononuclear cells (PBMC) cultured invitro.METHODS Epstein-Barr virus (EBV) was usedto transform the hepatitis C virus from a HCVpositive patient to permanent lymphoblastoidcell lines (LCL). Positive and negative HCV RNAstrands of the cultured cells and growth mediawere detected by reverse transcriptase-polymerase chain reaction ( RT-PCR ) eachmonth. Core and NS5 proteins of HCV werefurther tested using immunohistochemical SPmethod and in situ RT-PCR.RESULTS HCV RNA positive strands wereconsistently detected the cultured cells for oneyear. The negative-strand RNA in LCL cells andthe positive-strand RNA in supernatants wereobserved intermittently. Immunohistochemicalresults medicated expression of HCV NS3 and Cproteins in LCL cytoplasm mostly. The positivesignal of PCR product was dark blue and mainlylocalized to the LCL cytoplasm. The RT-PCRsignal was eliminated by overnight RNasedigestion but not DNase digestion.CONCLUSION HCV may exist and remainfunctional in a cultured cell line for a longperiod.展开更多
AIM:To investigate effects of iron on oxidative stress, heme oxygenase-1(HMOX1)and hepatitis C viral(HCV) expression in human hepatoma cells stably expressing HCV proteins. METHODS:Effects of iron on oxidative stress,...AIM:To investigate effects of iron on oxidative stress, heme oxygenase-1(HMOX1)and hepatitis C viral(HCV) expression in human hepatoma cells stably expressing HCV proteins. METHODS:Effects of iron on oxidative stress,HMOX1, and HCV expression were assessed in CON1 cells. Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction,and protein levels by Western blots. RESULTS:Iron,in the form of ferric nitrilotriacetate,increased oxidative stress and up-regulated HMOX1 gene expression.Iron did not affect mRNA or protein levels of Bach1,a repressor of HMOX1.Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2(Nrf2)by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels.These iron effects were completely blocked by deferoxamine(DFO).Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%, nonstructural 5A mRNA by 90%and protein by about 50%in the Con1 full length HCV replicon cells, whereas DFO increased them. CONCLUSION:Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells.This probably mitigates liver injury caused by combined iron overload and HCV infection.展开更多
Hepatitis C virus(HCV)is a major cause of liver disease worldwide.HCV is able to evade host defense mechanisms,including both innate and acquired immune responses,to establish persistent infection,which results in a b...Hepatitis C virus(HCV)is a major cause of liver disease worldwide.HCV is able to evade host defense mechanisms,including both innate and acquired immune responses,to establish persistent infection,which results in a broad spectrum of pathogenicity,such as lipid and glucose metabolism disorders and hepatocellular carcinoma development.The HCV genome is characterized by a high degree of genetic diversity,which can be associated with viral sensitivity or resistance(reflected by different virological responses)to interferon(IFN)-based therapy.In this regard,it is of importance to note that polymorphisms in certain HCV genomic regions have shown a close correlation with treatment outcome.In particular,among the HCV proteins,the core and nonstructural proteins(NS)5A have been extensively studied for their correlation with responses to IFN-based treatment.This review aims to cover updated information on the impact of major HCV genetic factors,including HCV genotype,mutations in amino acids 70 and91 of the core protein and sequence heterogeneity in the IFN sensitivity-determining region and IFN/ribavirin resistance-determining region of NS5A,on virological responses to IFN-based therapy.展开更多
AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of...AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.展开更多
Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)a...Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.展开更多
AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV...AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.展开更多
The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10...The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.展开更多
AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice.METHODS: The gene encoding the structural protein...AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice.METHODS: The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 μg.Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected.RESULTS: After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23(3.1±0.49) was higher than that in the control group (0.787±0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG.CONCLUSION: DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.展开更多
Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified a...Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.展开更多
Hepatitis C virus(HCV)helicase is a molecular motor that splits nucleic acid duplex structures during viral replication,therefore representing a promising target for antiviral treatment.Hence,a detailed understanding ...Hepatitis C virus(HCV)helicase is a molecular motor that splits nucleic acid duplex structures during viral replication,therefore representing a promising target for antiviral treatment.Hence,a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity.Despite extensive investigations performed in the past,a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved.Here we review investigations that have been previously performed by us for HCV helicase.Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation.We also discuss observations from that study in the light of recent experimental studies that confirm our findings.展开更多
In addition to causing cirrhosis and hepatocellular carcinoma, hepatitis C virus(HCV) is thought to cause hypolipidemia, hepatic steatosis, insulin resistance, metabolic syndrome, and diabetes. The viral life cycle of...In addition to causing cirrhosis and hepatocellular carcinoma, hepatitis C virus(HCV) is thought to cause hypolipidemia, hepatic steatosis, insulin resistance, metabolic syndrome, and diabetes. The viral life cycle of HCV depends on cholesterol metabolism in host cells. HCV core protein and nonstructural protein 5A perturb crucial lipid and glucose pathways, such as the sterol regulatory element-binding protein pathway and t he prot e in kinase B /mammal ian t arget of rapamycin/S6 kinase 1 pathway. Although several lines of transgenic mice expressing core or full HCV proteins exhibit hepatic steatosis and/or dyslipidemia, whether they completely reflect the metabolic alterations in humans with HCV infection remains unknown. Many cross-sectional studies have demonstrated increased prevalences of metabolic alterations and cardiovascular events in patients with chronic hepatitis C(CHC); however, conflicting results exist, primarily due to unavoidable individual variations. Utilizing anti-HCV therapy, most longitudinal cohort studies of CHC patients have demonstrated the favorable effects of viral clearance in attenuating metabolic alterations and cardiovascular risks. To determine the risks of HCV-associated metabolic alterations and associated complications in patients with CHC, it is necessary to adjust for crucial confounders, such as HCV genotype and host baseline glucose metabolism, for a long follow-up period after anti-HCV treatment. Adipose tissue is an important endocrine organ due to its release of adipocytokines, which regulate lipid and glucose metabolism. However, most data on HCV infection and adipocytokine alteration are inconclusive. A comprehensive overview of HCV-associated metabolic and adipocytokine alterations, from bench to bedside, is presented in this topic highlight.展开更多
To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) thro...To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage.展开更多
基金Supported by the National Natural Science Foundation of China for Distinguished Young Scholars, No. 30225040 for Dr Ning Q,No. 30123019 for Dr Luo XP
文摘AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinaseactivity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice.In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2)was detected in 21 of 23 patients (91.30%)with severe AOC hepatitis B, while only 1 of 13 patients(7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1of 10 with mild chronic hepatitis B had detectable hfgl2expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2protein play a pivotal role in initiating severe hepatitis.The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.
文摘Hepatitis C virus(HCV)infection is the leading cause of chronic liver-related diseases,including cirrhosis,liver failure,and hepatocellular carcinoma.Currently,no effective vaccine is available for HCV infection.Polyethylene glycol interferon-α(PegIFN-α)in combination with ribavirin(RBV)is the standard of care(SOC)for chronic hepatitis C.However,the efficacy of PegIFN-αand RBV combination therapy is less than 50%for genotype 1HCV,which is the dominant virus in humans.In addition,IFN and RBV have several severe side effects.Therefore,strategies to improve sustained virological response(SVR)rates have been an important focus for clinical physicians.The serine protease inhibitors telaprevir and boceprevir were approved by the United States Food and Drug Administration in 2011.The addition of HCV protease inhibitors to the SOC has significantly improved the efficacy of treatments for HCV infection.Several direct-acting antiviral drugs currently in late-stage clinical trials,both with and without pegIFN and RBV,have several advantages over the previous SOC,including higher specificity and efficacy,fewer side effects,and the ability to be administered orally,and might be optimal regimens in the future.Factors affecting the efficacy of anti-HCV treatments based on IFN-αinclude the HCV genotype,baseline viral load,virological response during treatment,host IL28B gene polymorphisms and hepatic steatosis.However,determining the effect of the above factors on DAA therapy is necessary.In this review,we summarize the development of antiHCV agents and assess the main factors affecting the efficacy of antiviral treatments.
基金Supported by National 863 Project,No.102-07-02-079th Five-Year Sci-Tech Plan,No.96-906A-03-08
文摘AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV gene fragments from core to NS3 and NS5 were constructed using prokaryotic expression vector pRSET and (or)pGEX. and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.``RESULTS One clone with HGV fragment from core to El(Gl). one from E2 (G31), three from NS3 (G6, G61, G7),one from NS5B (G821) and one chimeric fragment from NS3and NS5B (G61 821) could be expressed well and showed obvious immunoreactivity by Western blotting.One clone with I-KGV framment from NS5B (G82) was also well expressed, but could not show immunoreactivity by Western blotting. No obvious expression was found in the other six clones. All the expressed recombinant proteins were in inclusion body form, except the protein G61 which could be expressed in soluble form. Further purified recombinant proteins Gl, G,31, G61, G821 and G61 821were detected in indirected ELISA as coating antigen respectively. Only recombinant Gl could still show immunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivity of these four recombinant proteins were lost during purification.``CONCLUSION Core to El, E2. NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. A high. yield recombinant protein (Gl) located in HGV core to E1 could remain its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibody diagnosis.
基金This project was supported by grants from the Chinese National Thirteenth Five Years Project in Science and Technology (No. 2017ZX10202201), and Hubei Provincial Natural Science Foundation of China (No. 2018CFB206).
文摘The mechanisms involved in virus-induced severe hepatitis have not been fully elucidated. In this study, we investigated the role of gamma delta T cell receptors (γδ) T cells in the pathogenesis of fulminant viral hepatitis (FVH) induced by murine hepatitis virus strain 3 (MHV-3). The model of FVH was established by intraperitoneal injection of MHV-3 into Balb/cJ mice. The survival days of mice, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined. The proportions of γδ T cells in blood, spleen and liver, and cytokines secreted by hepatic γδ T cells were analyzed by flow cytometry. The function of hepatic γδ T cells was examined by cytotoxicity assay. Balb/cJ mice died in 3 to 6 days post MHV-3 infection, with severe hepatic necrosis and significant augmentation of serum ALT and AST levels. The proportions of γδ T cells in blood, spleen and liver were significantly increased post MHV-3 infection, while those of the early activating molecule CD69-expressing γδ T cells and productions of cytokines tumor necrosis factor-alpha (TNF-α) and interferon-y (IFN-3,) increased remarkably in the liver. These highly activated liver γδ T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect of liver γδ T cells against hepatocytes might involve the TNF-α and IFN-γ pathway. These results demonstrated that γδ T cells might contribute to the pathogenesis of MHV-3-induced FVH through the effector cytokines TNF-α and IFN-γ. Key words: fulminant viral hepatitis; murine hepatitis virus strain 3; gamma delta T cell receptors T cells; tumor necrosis factor-a; interferon-α
文摘AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected with HCV or transientlytransfected with a vector expressing HCV NS5 A. The effect of HCV NS5 A on the status of the critical players involved in insulin signaling was analyzed using realtime quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0.RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway,we analyzed the status of insulin receptor substrate-1(IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5 A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1(Ser307) in HCV infected or NS5 A transfected Huh7.5 cells compared to their respective controls. Furthermore,an increased phosphorylation of Akt(Ser473) was observed in HCV infected and NS5 A transfected cells compared to their mock infected cells. In contrast,we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5 A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5 A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue(Ser307) followed by decreased phosphorylation of Akt Thr308,Fox01 Ser256 and GSK3β Ser9,the downstream players of the insulin signalingpathway. Furthermore,increased expression of PECK and glucose-6-phosphatase,the molecules involved in gluconeogenesis,in HCV NS5 A transfected cells was observed.CONCLUSION: Taken together,our results suggest the role of HCV NS5 A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.
文摘The hepatitis C virus(HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases:(1) binding and internalization;(2) cytoplasmic release and uncoating;(3) viral polyprotein translation and processing;(4) RNA genome replication;(5) encapsidation(packaging) and assembly; and(6) virus morphogenesis(maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.
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The paper was support by a grant from the Ministry Youth Research of China,No.98-1-269
文摘AIM To study persistence and replication ofheltitis C virus (HCV) in patients' peripheralblood mononuclear cells (PBMC) cultured invitro.METHODS Epstein-Barr virus (EBV) was usedto transform the hepatitis C virus from a HCVpositive patient to permanent lymphoblastoidcell lines (LCL). Positive and negative HCV RNAstrands of the cultured cells and growth mediawere detected by reverse transcriptase-polymerase chain reaction ( RT-PCR ) eachmonth. Core and NS5 proteins of HCV werefurther tested using immunohistochemical SPmethod and in situ RT-PCR.RESULTS HCV RNA positive strands wereconsistently detected the cultured cells for oneyear. The negative-strand RNA in LCL cells andthe positive-strand RNA in supernatants wereobserved intermittently. Immunohistochemicalresults medicated expression of HCV NS3 and Cproteins in LCL cytoplasm mostly. The positivesignal of PCR product was dark blue and mainlylocalized to the LCL cytoplasm. The RT-PCRsignal was eliminated by overnight RNasedigestion but not DNase digestion.CONCLUSION HCV may exist and remainfunctional in a cultured cell line for a longperiod.
基金Supported by Grant(DK RO1 38825) and contracts(DK NO129236 and UO1 DK 06193)from the National Institutes of Health(NIDDK)
文摘AIM:To investigate effects of iron on oxidative stress, heme oxygenase-1(HMOX1)and hepatitis C viral(HCV) expression in human hepatoma cells stably expressing HCV proteins. METHODS:Effects of iron on oxidative stress,HMOX1, and HCV expression were assessed in CON1 cells. Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction,and protein levels by Western blots. RESULTS:Iron,in the form of ferric nitrilotriacetate,increased oxidative stress and up-regulated HMOX1 gene expression.Iron did not affect mRNA or protein levels of Bach1,a repressor of HMOX1.Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2(Nrf2)by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels.These iron effects were completely blocked by deferoxamine(DFO).Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%, nonstructural 5A mRNA by 90%and protein by about 50%in the Con1 full length HCV replicon cells, whereas DFO increased them. CONCLUSION:Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells.This probably mitigates liver injury caused by combined iron overload and HCV infection.
基金Supported by A Health and Labour Sciences Research Grant from the Ministry of Health,Labour and Welfare,Japan,a SATREPS Grant from Japan Science and Technology Agency and Japan International Cooperation Agencythe Japan Initiative for Global Research Network on Infectious Diseases(J-GRID)program from the Ministry of Education,Culture,Sports,Science and Technology,Japan
文摘Hepatitis C virus(HCV)is a major cause of liver disease worldwide.HCV is able to evade host defense mechanisms,including both innate and acquired immune responses,to establish persistent infection,which results in a broad spectrum of pathogenicity,such as lipid and glucose metabolism disorders and hepatocellular carcinoma development.The HCV genome is characterized by a high degree of genetic diversity,which can be associated with viral sensitivity or resistance(reflected by different virological responses)to interferon(IFN)-based therapy.In this regard,it is of importance to note that polymorphisms in certain HCV genomic regions have shown a close correlation with treatment outcome.In particular,among the HCV proteins,the core and nonstructural proteins(NS)5A have been extensively studied for their correlation with responses to IFN-based treatment.This review aims to cover updated information on the impact of major HCV genetic factors,including HCV genotype,mutations in amino acids 70 and91 of the core protein and sequence heterogeneity in the IFN sensitivity-determining region and IFN/ribavirin resistance-determining region of NS5A,on virological responses to IFN-based therapy.
基金Supported by National Natural Science Foundation of ChinaNo. 39670671, No. 30471531
文摘AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.
基金Supported by The National Institutes of Health,NO.DK080812
文摘Approximately 170 million people worldwide are chronically infected with hepatitis C virus(HCV).Chronic HCV infection is the leading cause for the development of liver fibrosis,cirrhosis,hepatocellular carcinoma(HCC)and is the primary cause for liver transplantation in the western world.Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of typeⅡdiabetes.Insulin resistance plays an important role in the development of various complications associated with HCV infection.Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis,steatosis,HCC and resistance to anti-viral treatment.Thus,HCV associated insulin resistance is a therapeutic target at any stage of HCV infection.HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway.Various mechanisms have been proposed in regard to HCV mediated insulin resistance,involving up regulation of inflammatory cytokines,like tumor necrosis factor-α,phosphorylation of insulin-receptor substrate-1,Akt,up-regulation of gluconeogenic genes like glucose 6 phosphatase,phosphoenolpyruvate carboxykinase 2,and accumulation of lipid droplets.In this review,we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.
基金the Universidad de Buenos Aires(SECyT-UBA,TB14)Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET,PIP723)+1 种基金Agencia Nacional de Promoción Científica y Tecnológica(ANPCyT,PICT 01610)Ministerio de Salud Pǘblica de la Nación(Beca Carrillo-Oativia)
文摘AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.
基金supported by the National Key Technology R&D Program of China (2015BAD12B01-2)the Major Program of National Natural Science Foundation of China (31490603)the earmarked fund for Modern Agroindustry Technology Research System of China (CARS-36)
文摘The nonstructural protein 10(nsp10) of porcine reproductive and respiratory syndrome virus(PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.
基金Supported by the Grants from the Natural Science Foundation of Zhejiang Province, No. RC01054, Science Technology Department of Zhejiang Province, No. F11023 and Key Project of Health Bureau of Zhejiang Province
文摘AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice.METHODS: The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 μg.Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected.RESULTS: After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23(3.1±0.49) was higher than that in the control group (0.787±0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG.CONCLUSION: DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice.
文摘Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.
文摘Hepatitis C virus(HCV)helicase is a molecular motor that splits nucleic acid duplex structures during viral replication,therefore representing a promising target for antiviral treatment.Hence,a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity.Despite extensive investigations performed in the past,a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved.Here we review investigations that have been previously performed by us for HCV helicase.Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation.We also discuss observations from that study in the light of recent experimental studies that confirm our findings.
基金Supported by Grants from the Chang Gung Medical Research ProgramNo.CMRPG380353+7 种基金No.CMRPG3B1251No.CMRPG3B0401No.CMRPG3B1741No.CMRPG3B1742 and No.XMRPG3A0521the National Science CouncilTaiwanNo.100-2314-B-182-069-No.101-2314-B-182-083-and No.102-2628-B-182-021-MY3
文摘In addition to causing cirrhosis and hepatocellular carcinoma, hepatitis C virus(HCV) is thought to cause hypolipidemia, hepatic steatosis, insulin resistance, metabolic syndrome, and diabetes. The viral life cycle of HCV depends on cholesterol metabolism in host cells. HCV core protein and nonstructural protein 5A perturb crucial lipid and glucose pathways, such as the sterol regulatory element-binding protein pathway and t he prot e in kinase B /mammal ian t arget of rapamycin/S6 kinase 1 pathway. Although several lines of transgenic mice expressing core or full HCV proteins exhibit hepatic steatosis and/or dyslipidemia, whether they completely reflect the metabolic alterations in humans with HCV infection remains unknown. Many cross-sectional studies have demonstrated increased prevalences of metabolic alterations and cardiovascular events in patients with chronic hepatitis C(CHC); however, conflicting results exist, primarily due to unavoidable individual variations. Utilizing anti-HCV therapy, most longitudinal cohort studies of CHC patients have demonstrated the favorable effects of viral clearance in attenuating metabolic alterations and cardiovascular risks. To determine the risks of HCV-associated metabolic alterations and associated complications in patients with CHC, it is necessary to adjust for crucial confounders, such as HCV genotype and host baseline glucose metabolism, for a long follow-up period after anti-HCV treatment. Adipose tissue is an important endocrine organ due to its release of adipocytokines, which regulate lipid and glucose metabolism. However, most data on HCV infection and adipocytokine alteration are inconclusive. A comprehensive overview of HCV-associated metabolic and adipocytokine alterations, from bench to bedside, is presented in this topic highlight.
基金National 973 project of Chnia for SARS study (2003CB514112) Ministry of Education of China for SARS study (2003-18) National Science Fund for Distinguished Young Investigators (30225040,. 30123019).
文摘To evaluate the role of murine fibrinogen like protein 2 (mfgl2) /fibroleukin in lung impairment in Severe acute respiratory syndrome (SARS), a murine SARS model induced by Murine hepatitis virus strain 3 (MHV-3) through trachea was established. Impressively, all the animals developed interstitial pneumonia with extensive hyaline membranes formation within alveoli, and presence of micro-vascular thrombosis in the pulmonary vessels. MHV-3 nucleocapsid gene transcripts were identified in multiple organs including lungs, spleen etc. As a representative proinflammatory gene, mfgl2 prothrombinase expression was evident in terminal and respiratory bronchioles, alveolar epithelia and infiltrated cells in the lungs associated with fibrin deposition and micro-vascular thrombosis. In summary, the established murine SARS model could mimic the pathologic characteristics of lungs in patients with SARS. Besides the physical damages due to virus replication in organs, the up-regulation of novel gene mfgl2 in lungs may play a vital role in the development of SARS associated lung damage.