BACKGROUNDHepatitis C virus (HCV) core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase (PWP). For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis.

METHODSSamples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study.

RESULTSOnly 48 (1.3%) of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 (24.3%) of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 (60%) were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% (20/23) and 69.6% (16/23) respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test (r = -0.989, P < 0.05), and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV titres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually.

CONCLUSIONSThe HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.

Alanine Transaminase ; blood ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; RNA, Viral ; blood ; Sensitivity and Specificity ; Viral Nonstructural Proteins ; blood

BACKGROUNDTo determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli.

METHODSHGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified.

RESULTSAfter identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens.

CONCLUSIONSNS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

Antibodies, Viral ; blood ; Antigens, Viral ; blood ; Epitopes ; immunology ; GB virus C ; genetics ; immunology ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

Interferon-alpha (IFN-alpha) has been used to treat hepatitis C virus (HCV)-induced hepatitis, but it has been effective in only about half of the treated patients, with recurrence appearing in the other half. As a consequence of the possible complications associated with IFN-alpha and the high cost of treatment, it has become extremely important to select the proper patients for IFN-alpha treatment. In our previous study, we found that the quasispecies in the hypervariable region (HVR) 1 of HCV were various and that a new quasispecies can appear in non-responders and/or lead to deterioration in the patients' condition. The preliminary data we obtained in the process of our previous research led us to believe that the quasispecies of HVR 1 has something to do with the effect of IFN-alpha. Thus, in this investigation, we tried to determine the predictive factors of IFN-alpha therapy. Thirty patients with HCV infection were treated with IFN-alpha. Among them, 15 patients recovered after six months IFN-alpha treatment, but the remaining 15 patients showed no response after six months IFN-alpha treatment. We cloned HVR 1 DNA by reverse transcription-polymerase chain reaction (RT-PCR) and examined the quasispecies of HVR 1. As the quasispecies of HVR 1 in non-responders varied more than in the complete remission group, we concluded that the sequence variation in HVR 1 of HCV can be used to predict the effect of IFN-alpha.
Adult ; Aged ; Amino Acid Sequence ; Female ; Genotype ; Hepatitis C/virology ; Hepatitis C/drug therapy* ; Hepatitis C-Like Viruses/classification* ; Human ; Interferon-alpha/therapeutic use* ; Male ; Middle Age ; Molecular Sequence Data ; Viral Nonstructural Proteins/genetics ; Viral Nonstructural Proteins/chemistry* ; Viral Nonstructural Proteins/blood

Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E + prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.
Animals ; Antibodies, Viral/blood ; Baculoviridae/genetics ; Blotting, Western ; Cloning, Molecular ; Encephalitis Virus, Japanese/genetics/*immunology ; Encephalitis, Japanese/*immunology/prevention&control ; Female ; Immunization ; Immunoglobulin Isotypes/blood ; Japanese Encephalitis Vaccines/*immunology/standards ; Mice ; Mice, Inbred ICR ; Microscopy, Fluorescence ; Plasmids ; Recombinant Proteins/genetics/immunology ; Viral Envelope Proteins/genetics/*immunology ; Viral Matrix Proteins/genetics/*immunology ; Viral Nonstructural Proteins/genetics/*immunology

BACKGROUNDTo elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy.

METHODSSera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken.

RESULTSPretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain.

CONCLUSIONSLow HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.

Amino Acid Sequence ; Antiviral Agents ; therapeutic use ; Hepacivirus ; drug effects ; genetics ; Hepatitis C, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; RNA, Viral ; blood ; Viral Nonstructural Proteins ; genetics

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.
Adult ; Aged ; Female ; Genotype ; Hepatitis C/immunology* ; Hepatitis C/drug therapy* ; Hepatitis C/diagnosis ; Hepatitis C/blood ; Hepatitis C Antibodies/immunology* ; Hepatitis C Antibodies/blood ; Hepatitis C Antigens/immunology* ; Hepatitis C-Like Viruses/immunology* ; Hepatitis C-Like Viruses/genetics ; Human ; Immunoblotting ; Interferon Alfa-2a/therapeutic use* ; Male ; Middle Age ; RNA, Viral/blood ; Recombinant Fusion Proteins/immunology ; Viral Core Proteins/immunology* ; Viral Nonstructural Proteins/immunology*

BACKGROUND: Although the polyproteins of hepatitis C virus(HCV) are processed and formed in nearly equimolar amounts, individual functional proteins have a discrepancy in their time of appearance following HCV infection and eliciting immune response. This study was conducted to compare the reactivity toward regional specific HCV protein in relation to virological characteristics, including HCV genotype and HCV replication. METHODS: Sera from forty-five patients with chronic HCV infection were analyzed through the experiments of the recombinant immunoblot assay(RIBA-2), HCV genotyping and HCV RNA quantitation. RESULTS: The frequencies of seropositivity to C22-3, C33C, C100-3 and 5-1-1 proteins were 91.1+ACU-, 91.1+ACU-, 64.4+ACU- and 53.3+ACU-, respectively, of all the patients, and thus the antibodies to C22-3 and C33C proteins were found more frequently (p +ADw- 0.05). The antibody responses between core or NS3 proteins and NS4 proteins showed more discrepancy in the HCC group than that in the CH group, implying a possibility of oncogenic potential of core or NS3 gene in hepatocarcinogenesis. The detection rate of antibodies to C22-3 and C33C, in accordance with serum HCV RNA levels, was significantly higher in highly viremic patients than that in low viremic patients (p +ADw- 0.05). Antibodies to C22-3, C33C, C100-3 and 5-1-1 were also found more frequently in patients with HCV genotype 1b, compared to those with HCV genotype 2a (p +ADw- 0.05). CONCLUSION: These results suggest that antibody detection of HCV may depend on the virological characteristics of HCV, the levels of HCV replication and HCV genotype and, therefore, HCV RNA detection using RT-PCR technique is essential for confirmatory diagnosis for HCV infection. Furthermore, the HCV core or NS3 Protein may play important role in hepatocarcinogenesis.
Adult ; Aged ; Female ; Genotype ; Hepatitis C Antibodies/blood+ACo- ; Hepatitis C, Chronic/virology ; Hepatitis C, Chronic/immunology+ACo- ; Hepatitis C-Like Viruses/physiology ; Hepatitis C-Like Viruses/genetics ; Human ; Male ; Middle Age ; RNA, Viral/blood ; Viral Core Proteins/immunology+ACo- ; Viral Nonstructural Proteins/immunology+ACo- ; Virus Replication

OBJECTIVETo identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene.

METHODSIn total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum.

RESULTSThe sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera.

CONCLUSIONThe major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

Amino Acid Sequence ; Antigenic Variation ; Antigens, Viral ; blood ; immunology ; Epitope Mapping ; Genotype ; Hepacivirus ; classification ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Hepatitis C Antibodies ; blood ; immunology ; Humans ; Immunodominant Epitopes ; Peptides ; chemical synthesis ; immunology ; Recombinant Proteins ; genetics ; immunology ; Sequence Homology, Amino Acid ; Viral Nonstructural Proteins ; genetics ; immunology

OBJECTIVETo study cellular and humoral immune responses to NS1 protein in mice inoculated intramuscularly with recombinant plasmid expressing dengue 2 NS1 gene.

METHODSThe eukaryotic expressing plasmid pCNX2-NS1 was injected into tibialis anterior muscle in mice. The mice were subsequently boosted with the same dose and same method twice after the initial inoculation. The mice were killed at four-week intervals and their serum and spleen cells were harvested for further test.

RESULTSDengue 2 antibodies were detectable in the sera from inoculated animals four weeks after the last boost. The changes of CD4+ T lymphocyte and CD8+ T lymphocyte were also determined by flow cytometry.

CONCLUSIONThe recombinant plasmid containing dengue 2 NS1 genes is immunogenic in intramuscularly inoculated mice. The vaccinated mice produced dengue-2 specific and long lasting immunity.

Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Dengue ; blood ; immunology ; virology ; Dengue Vaccines ; immunology ; Dengue Virus ; genetics ; immunology ; Female ; Flow Cytometry ; Gene Expression ; Immunization ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Vaccines, Combined ; immunology ; Vaccines, DNA ; genetics ; immunology ; Viral Nonstructural Proteins ; genetics