Cloning and identification of NS5A TP2gene and its spliced variant transactivated by hepatitis C virus non-structural protein 5A

AIM:To clone,identify and study new NS5ATP2 gene andits spliced variant transactivated by hepatitis C virus non-structural protein 5A.METHODS:On the basis of subtractive cDNA library of genestransactivated by NS5A protein of hepatitis C virus,the codingsequence of new gene and its spliced variant were obtainedby bioinformatics method.Polymerase chain reaction(PCR)was conducted to amplify NS5ATP2 gene.RESULTS:The coding sequence of a new gene and itsspliced variant were cloned and identified successfully.CONCLUSION:A new gene has been recognized as thenew target transactivated by HCV NS5A protein.These resultsbrought some new clues for studying the biological functionsof new genes and pathogenesis of the viral proteins.     

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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Role of hepatitis C virus in myocarditis and cardiomyopathies

Recent nationwide clinico-epidemiological surveys in Japan showed that the occurrence of cardiomyop-athies was most frequently seen in the age of sixties,and that cardiomyopathies are important causes of heart failure inthe elderly.Viral infection was conventionally considered to cause myocarditis,which resulted in the development ofdilated cardiomyopathy.Recent studies suggest that hepatitis C virus(HCV)is involved in the development of dilatedcardiomyopathy,hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy in addition to myo-carditis.Furthermore,left ventricular aneurysm represents the same morbid state not only after myocardial infarctionbut also after myocarditis.There were wide variations in the frequency of detection of HCV genomes in cardiomyopathyin different regions and in different populations.Major histocompatibility complex class Ⅱ genes may play a role in thesusceptibility to HCV infection,and may influence the development of different phenotypes of cardiomyopathy.If infact the myocardial damage is caused by HCV,it might be expected that interferon(IFN)administration would beuseful for its treatment.Hepatitis patients receiving IFN treatment for hepatitis were screened by thallium myocardialscintigraphy,and an abnormality was discovered in half of the patients.Treatment with IFN resulted in a disappear-ance of the image abnormality.It has thus been suggested that mild myocarditis and myocardial damage may be curedwith IFN.We have recently found that high concentrations of circulating cardiac troponin T are a specific marker ofcardiac involvement in HCV infection.By measuring cardiac troponin T in patients with HCV infection,the preva-lence of cardiac involvement in HCV infection will be clarified.We are proposing a collaborative work on a globalnetwork on myocarditis/cardiomyopathies due to HCV infection.(J Geriatr Cardiol 2004;1(2):83-89.)     

Deletion of protein kinase Cε in mice has limited effects on liver metabolite levels but alters fasting ketogenesis and gluconeogenesis

Aims/hypothesis  

Protein kinase Cε (PKCε) is emerging as a key mediator of lipid-induced insulin resistance in liver and hepatic lipid metabolism itself. We investigated whether PKCε plays a role in other metabolic processes, to further examine its suitability as a therapeutic target.     

Occult hepatitis C virus infection： A new form of hepatitis C

INTRODUCTION The etiology of liver disease is unknown in approximately 10% of patients with abnormal results on liver function tests. Some authors have reported that occult hepatitis B virus could be the cause of a proportion of these cryptogenic chronic …     

Inhibitory effect of oxymatrine on serum hepatitis B virus DNA in HBV transgenic mice

AIM:To study the inhibitory effect of oxymatrine on serumhepatitis B virus (HBV) DNA in HBV transgenic mice.METHODS:HBV transgenic mice model was establishedby microinjection,and identified by HBV DNA integrationand replication.Transgenic mice with replicating HBV weredivided into 3 groups,and injected with normal saline(group A,n=9),50 mglkg (group B,n=8) and 100 mg/kg(group C,n=9) oxymatrine intraperitoneally once a dayfor 30d,respectively.Quantitation of serum HBV DNA inHBV transgenic mice was performed by competitivepolymerase chain reaction (PCR) in combination with DNAhybridization quantitative detection technique before andafter treatment.RESULTS:Compared with pre-treatment,the serum HBVDNA in group A (F=1.04,P=0.9612) and group B (F=1.13,P=0.8739) had no changes after treatment.However,in groupC serum HBV DNA was significantly decreased (F=13.97,P=0.0012).The serum HBV DNA after treatment was lowerin group C than in groups B and A (F=8.65,P=0.0068;F=12.35,P=0.0018;respectively).The serum HBV DNAafter treatment was lower in group B than in group A,butthere was no statistical significance (F=1.43,P=0.652).CONCLUSION:Oxymatrine has inhibitory effects on serumHBV DNA in HBV transgenic mice.     

Immunogenicity of recombinant hepatitis B virus vaccine in patients with and without chronic hepatitis C virus infection： A case-control study

AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.     

Transactivating effect of hepatitis C virus core protein： A suppression subtractive hybridization study

AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein. METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BarnHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cell swere transiently transfected with pcDNA3.1(-)-core using Upofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coil strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR). RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of β-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by Genl3ank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein. CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.     

Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells

AIM:To investigate the effects of hepatitis B virus×geneand its protein product HB×Ag on apoptosis in hepatocyteline HL-7702.METHODS:The reconstituted plasmid pcDNA3-x wasestablished through recombination DNA technique;pcDNA3-X was transfected into HL-7702 cells by lipid-mediatedtrasfection.Positive clones were screened by G418,and HL-7702/HBx cells were analysed by the RT-PCR to confirm thesteady expression of X gene in HL-7702 cells.The apoptosisrate in HL-7702 cells was determined by flow cytometry,TUNEL technology,electronic microscope.At the mean time,pcDNA3-X was transfected transiently into HL-7702 cells,and total RNA from HL-7702 cells was extracted 24,48,72,96 and 120 h after the transient transfection,and semi-quantitative analysis was performed by RT-PCR to detectthe expression of HBV X gene.Furthermore,apoptosis ratein HL-7702 cells was determined by flow cytometry analysisat the different times.RESULTS:RT-PCR analysis showed that HBV X gene couldbe expressed stably in HL-7702 cells.Both flow cytometryand TUNEL technology revealed that the apoptosis rates ofHL-7702/HBx cells were much higher than those of HL-7702/pcDNA3 and HL-7702 cells.Furthermore,the apoptoticphenomena and apoptotic body were observed in HL-7702/HBx cells under electronic microscope,but not in HL-7702/pcDNA3 and HL-7702 cells.In the experiment of transienttransfection,RT-PCR reveals that X gene was expressed mostat 72 h after transfection;and the apoptosis rate reachedthe highest at the same time.After that,the apoptosis ratewas reduced with the decrease of the X gene expression.CONCLUSION:HBV X gene and X protein can promote theapoptosis in hepatocyte.And there exist a quantity-effectrelationship between the X gene expression and apoptosisrate in hepatocyte.     

Recent advances in hepatitis C virus research and understanding the biology of the virus

Since the identification of hepatitis C virus (HCV) genome in 1989[1], a lot of progresses have been done about the understanding of HCV biology, natural history and therapeutic options. HCV is a member of the Flaviviridae viral family. Its genome is a positive simple strand RNA     

Transactivating effect of hepatitis C virus core protein: A suppression subtractive hybridization study

AIM:To investigate the transactivating effect of hepatitis Cvirus(HCV)core protein and to screen genes transactivatedby HCV core protein.METHODS:pcDNA3.1(-)-core containing full-length HCVcore gene was constructed by insertion of HCV core geneinto EcoRI/BamHI site.HepG2 cells were cotransfected withpcDNA3.1(-)-core and pSV-lacZ.After 48 h,cells werecollected and detected for the expression of β-gal by anenzyme-linked immunosorbent assay(ELISA)kit.HepG2 cellswere transiently transfected with pcDNA3.1(-)-core usingLipofectamine reagent.Cells were collected and total mRNAwas isolated.A subtracted cDNA library was generated andconstructed into a pGEM-Teasy vector.The library wasamplified with E.coli strain JM109.The cDNAs weresequenced and analyzed in GenBank with BLAST search afterpolymerase chain reaction(PCR).RESULTS:The core mRNA and protein could be detected inHepG2 cell lysate which was transfected by the pcDNA3.1(-)-core.The activity of β-galactosidase in HepG2 cells transfectedby the pcDNA3.1(-)-core was 5.4 times higher than that ofHepG2 cells transfected by control plasmid.The subtractivelibrary of genes transactivated by HCV core protein wasconstructed successfully.The amplified library contained 233positive clones.Colony PCR showed that 213 clonescontained 100-1000 bp inserts.Sequence analysis wasperformed in 63 clones.Six of the sequences were unknowngenes.The full length sequences were obtained withbioinformatics method,accepted by GenBank.It wassuggested that six novel cDNA sequences might be targetgenes transactivated by HCV core protein.CONCLUSION:The core protein of HCV has transactivatingeffects on SV40 early promoter/enhancer.A total of 63 clonesfrom cDNA library were randomly chosen and sequenced.Using the BLAST program at the National Center forBiotechnology Information,six of the sequences wereunknown genes.The other 57 sequences were highly similarto known genes.     

Effect of SEN virus coinfection on outcome of lamivudine therapy in patients with hepatitis B

AIM:Interactions between hepatitis B virus (HBV) and otherviral hepatitis infections are well known,whether the newlydiscovered SEN virus (SENV) has any effect on lamivudineantiHBV activity is unclear.Our aim was to clarify the effecton treatment outcome of coinfection with SEN virus inpatients with hepatitis B during lamivudine therapy.METHODS:Nested polymerase chain reaction (PCR)amplification was used to detect SENV-D and SENV-Hstrains in serum from 45 patients with chronic hepatitis Btreated with lamivudine 100 mg daily for 12 mo.HBV DNAload was detected with fluorescence quantitative PCR (FQ-PCR) and YMDD (tyrosine,methionine,aspartate,aspartate)motif mutation of HBV DNA was investigated with cDNAmicroarray.RESULTS:SENV DNA was detected in 5 of 45(11.1%) casesafter 12 mo they received lamivudine treatment.SENV-Dand SENV-H were 4.4% and 6.7% respectively.HBV DNAfailed to respond to lamivudine therapy in 4 of 5 SENVcoinfected patients while only 10 of 40 patients becameSENV positive and the difference was statistically significant.Response of ALT and HBeAg to lamivudine had no significantdifference between coinfection patients and single HBVinfection ones.CONCLUSION:Coinfection with SEN virus in chronichepatitis B patients may adversely affect the outcome oflamivudine treatment.     

Is the hepatitis C virus epidemic over in Egypt? Incidence and risk factors of new hepatitis C virus infections

Objectives: To estimate hepatitis C virus (HCV) incidence rates and identify risk factors for current HCV transmission with emphasis on the role of living with infected household family members in rural Egypt. Methods: A 4‐year population‐based, cohort study of seronegative villagers was conducted to identify incident HCV seroconversion cases. A risk factor questionnaire and blood samples for anti‐HCV EIA‐3 and HCV RNA polymerase chain reaction testing were collected at two rounds of follow‐up. Incidence rates, relative risks and 95% confidence interval (CI) were calculated based on a Poisson distribution. A matched case–control analysis to explore specific behavioural predictors of infection was conducted and odds ratios were obtained by conditional logistic regression. Results: Twenty‐five participants (11 females) seroconverted in 10 578 person years of follow‐up (PY), (incidence rate of 2.4/1000 PY; 95% CI: 1.6–3.5). The median age at seroconversion was 26 years [interquartile range (IQR) 19–35] among males and 20 years (IQR 13–24) among females. The only significant risk factor identified for these cases was receiving injections [adjusted odds ratio (OR_adj)=3.3; 95% CI: 1.1–9.8]. Two of the 17 viraemic seroconvertors were infected with the same strain as at least one of their family members. Conclusion: This study identified the important role of injections in spreading HCV infection in this rural community. National healthcare awareness and infection control programmes should be strengthened to prevent further transmission. Screening of families of infected HCV subjects should be an essential part of case management for early detection and management.     

Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

AIM:To explore the role of hepatitis C virus(HCV)envelopeprotein 2(E2)in the induction of apoptosis.METHODS:A carboxyterminal truncated E2(E2-661)wastransiently expressed in several cultured mammalian celllines or stably expressed in Chinese hamster ovary(CHO)cell line.Cell proliferation was assessed by ~3H thymidineuptake.Apoptosis was examined by Hoechst 33258staining,flow cytometry and DNA fragmentation analysis.RESULTS:Reduced proliferation was readily observed inthe E2-661 expressing cells.These cells manifested thetypical features of apoptosis,including cell shrinkage,chromatin condensation and hypodiploid genomic DNAcontent.Similar apoptotic cell death was observed in anE2-661 stably expressing cell line.CONCLUSION:HCV E2 can induce apoptosis in culturedmammalian cells.     

Impact of human immunodeficiency virus infection on the course of hepatitis C virus infection： A meta-analysis

AIM： TO analyze the influence of human immunodeficiency virus （HIV） infection on the course of hepatitis C virus （HCV） infection. METHODS： We performed a meta-analysis to quantify the effect of HIV co-infection on progressive liver disease in patients with HCV infection. Published studies in the English or Chinese-language medical literature involving cohorts of HIV-negative and -positive patients coinfected with HCV were obtained by searching the PUBMED, EMBASE and CBM. Data were extracted independently from relevant studies by 2 investigators and used in a fixed-effect meta analysis to determine the difference in the course of HCV infection in the 2 groups. RESULTS： Twenty-nine trails involving 16750 patients were identified including the outcome of histological fibrosis or cirrhosis or de-compensated liver disease or hepatocellular carcinoma or death. These studies yielded a combined adjusted odds ratio （OR） of 3.40 [95% confidence interval （CI） = 2.45 and 4.73]. Of note, studies that examined histological fibrosis/ cirrhosis, decompensated liver disease, hepatocellular carcinoma or death had a pooled OR of 1.47 （95% CI = 1.27 and 1.70）, 5.45 （95% CI = 2.54 and 11.71）, 0.76 （95% CI = 0.50 and 1.14）, and 3.60 （95% CI = 3.12 and 4.15）, respectively. CONCLUSION： Without highly active antiretroviral therapies （HAART）, HIV accelerates HCV disease progression, including death, histological fibrosis/ cirrhosis and decompensated liver disease. However, the rate of hepatocellular carcinoma is similar in persons who had HCV infection and were positive for HIV or negative for HIV.     

A prospective study of vertical transmission of hepatitis C virus

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Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

AIM:To construct pEGFP-hepatocyte growth factor(HGF)expression vector,the to detect its expression in transfectedhuman hepatocytes,and to investigate the influence ofautocrine HGF expression on the proliferative potential andcytoprotective effects in human hepatocytes.METHODS:Human HGF cDNA was ligated to the pEGFP vector.Recombinant plasmid was transfected into human hepatocyteline QZG wibh liposome.Expression of HGF protein was observedby fluorescence microscopy and immunohistochemistry.Hepaticcells were collected 24,48,and 72 h after transfection todetect the number of[~3H]-TdR uptake in DNA.DNA synthesiswas observed by using PCNA stain immunohistochemistry.Acute liver cell damage was induced by carbontetrachloride.Cytoprotective effect was observed byexamining the survival rate of hepatocytes and leakage ofintracellular alanine transaminase(ALT)and potassium ions.RESULTS:HGF identification of pEGFP-HGF by enzymedigestion showed that HGF fragment was cloned into BamHI and Sa/I sites of pEGFP-N3.Expression of GFP in transfectedhepatocytes was observed with fluorescence microscopy.The[~3H]-TdR uptake became 7 times as many as in thecontrol group 96 h after transfection.After HGF transfection,the survival rate of hepatocytes poisoned by CCI_4 significantlyincreased(83% vs 61%,P<0.05),and the leakage ofintracellular alanine transaminase and potassium ions decreased(586 nkat/L vs 1089 nkat/L,P<0.01;and 5.59 mmol/L vs6.02 mmol/L,P<0.01 respectively).Culture of transfectedhepatic cells promoted the proliferation of other non-transfected cells.CONCLUSION:Transfected HGF is expressed in hepaticceils and has the activity of promoting cell division andprotecting hepatic cells against poisoning.