Hepatitis C Virus RNA-positive Sera Research Articles

Distribution of Hepatitis C Virus Genotypes in Kayseri Region, in Turkey: Unexpected Rate of Genotype 4

Hepatitis C virus (HCV) is an important cause of chronic liver disease. There are six genotypes and more than 80 subtypes of HCV. The aim of this study was to investigate the distribution of HCV genotypes in Middle Anatolia in Turkey, and the association of HCV genotypes with pre-treatment HCV RNA viral load, serum transaminase levels, and histopathological grade of liver fibrosis. A total of 160 patients (103 female, 57 male) with chronic hepatitis C were retrospectively evaluated. HCV RNA level was determined by commercial real time PCR method. HCV RNA positive sera were genotyped by the Abbott Real Time HCV Genotype II assay and sequenced by the ABI Prism 310 Genetic Analyzer. Gender, age, serum ALT, AST, HCV RNA viral load, and fibrosis staging of liver were determined in all patients. Genotype 1b was the most frequent (64.7%) followed by genotype 4d (28.3%), 2 (4.4%), and la (2.5%). The HCV genotype results were found consistent with both methods. The gender distribution of the 160 HCV infected patients was 57 male/103 female. Log HCVRNA was significantly higher in genotype 1b compared to genotype 4 and 1a. Stage of liver fibrosis, histology activity index, serum ALT and AST levels did not differ between groups depending on genotypes. Advanced liver fibrosis (Group 2) was found in 36 (76.6%) patients with genotype 1b and in 10 (21.3%) patients with genotype 4 and only in 1 patient (2.1%) with genotype 1a. HCV genotype 1b is the most frequent type (64.7%) in this region. Prevalence of genotype 4 in this region is higher than the national HCV genotype distribution. Serum transaminase levels and liver fibrosis scores are not associated with HCV genotypes.

Natural intragenotypic and intergenotypic HCV recombinants are rare in southwest China even among patients with multiple exposures

Background Both intragenotypic and intergenotypic hepatitis C virus (HCV) recombinants were recently identified, whereas the frequency of HCV recombinants in circulation in certain parts of the world remains uncertain. Objectives To assess the frequency of natural intragenotypic and intergenotypic HCV recombinants in southwest China and that of multitypic HCV infection, which is the prerequisite factor for recombination. Study design Comparison of the genotyping results based on core-envelope 1 and non-structural 5B sequence phylogenetic analyses to detect the possible recombinants. Multitypic HCV infection was detected by colony analysis of the core-envelope 1 region. Results There was no discrepancy between genotyping results based on the core-envelope 1 and non-structural 5B regions in 243 sera from 222 patients, including 107 samples from 92 intravenous drug users, 26 samples from 20 hemodialysis patients, and 110 samples from 110 other patients. Multitypic HCV carriage was identified in 9 (47.4%) of the 19 patients with multiple exposures who were randomly selected, and 11 (45.8%) of the 24 specimens. There were 26 repeatedly exposed patients who had more than one HCV RNA positive sera at different time points, but neither of their HCV subtypes had switched over time. Conclusion Although the high frequency of multitypic HCV infection was identified in patients with multiple exposures, intergenotypic or intragenotypic recombination remains an infrequent event in southwest China; thus, routine genotyping with more than one subgenomic region for clinical use is not warranted.

Antigenic structure of the hepatitis C virus envelope 2 protein

The antigenic structure of the envelope 2 (e2) protein of the hepatitis C virus (HCV) was characterized by the use of 70 synthetic peptides and 131 human sera from persons with antibodies to HCV. Among 34 overlapping peptides spanning the e2 protein of HCV, two major antigenic regions were located to residues 484-499 and residues 554-569. The sequence of the two major antigenic regions of the e2 protein are partly well conserved within the described types of HCV. Both regions contain two Cys residues in close proximity, and the region at residues 554-569 contains a putative N-glycosylation site, which are factors that previously have been suggested to affect the immune recognition of the e2 protein. Using substitution peptide analogues where each position within residues 484-499 and 554-569 were sequentially substituted by Ala or Gly, the most essential residues for antibody binding were found to be the Pro-498, Ala-499, Ala-566, Pro-567, and Pro-568. All of these, except for the Pro-498 and Ala-566, are conserved among different HCV strains. Also, according to previous studies, position 496 often shows variations, which could be explained by position 496 being contained within the antigenic region at residues 484-499. Interestingly, none of the Cys residues at positions 486, 494, 564 and 569 were found to be essential for antibody binding, indicating that these are not essential in maintaining the e2 antigenicity of the peptides. In a material of 114 confirmed anti-HCV positive sera, derived from patients during the acute or the chronic phase of HCV infection, the prevalence of antibodies to the two major linear antigenic regions of the e2 protein was found to be 55% among HCV RNA-positive sera, and 53% among HCV RNA-negative sera. In conclusion, we have identified and characterized two major linear antigenic regions outside the two hypervariable regions of the e2 protein. Since these regions are accessible to the B cells of the infected host, these two regions are likely to be surface exposed either on the precursor polyprotein or the native e2 protein. Also, we could confirm that antibodies to the e2 protein co-exist with HCV viraemia.

Hepatitis C virus infection in Jordanian haemodialysis units: serological diagnosis and genotyping.

The seroprevalence and genotypes of hepatitis C virus (HCV) were studied in 283 patients attending six haemodialysis units in Jordan. In all, 98 (34.6%) patients were anti-HCV-positive by EIA, 92 (93.9%) of whom were also reactive in an immunoblot assay. The prevalence of anti-HCV was correlated with a history of blood transfusion before the introduction of blood donor screening for HCV and with duration of haemodialysis. HCV RNA was detected in 30 (30.6%) of 98 anti-HCV-positive sera. HCV viraemia was not associated with a particular antibody for the six HCV antigens studied by the immunoblot assay, although reactivity to the core antigens was greater in the HCV RNA-positive sera than in negative sera. Two HCV genotypes (1 and 4) were identified for the first time in Jordan by restriction fragment length polymorphism analysis of HCV 5'-NCR. The predominant genotype was HCV la (12 of 30). Genotypes lb and 4 were detected in 10 and 8 patients, respectively. The antibody response to HCV antigens was genotype-dependent, with a wider range of antibody specificities detected in the immunoblot assay in the 12 patients with genotype 1a infection than in the 8 patients with genotype 4. However, there was no significant difference in the prevalence of antibodies to HCV antigens among patients infected with either genotype 1a or lb. In conclusion, the prevalence of anti-HCV, blood transfusion, duration of dialysis and HCV genotypes suggest possible nosocomial HCV transmission among patients which needs confirmation by phylogenetic analysis of subgenomic HCV regions.

Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinaemia.

The IgMk rheumatoid factors (RF) of type II mixed cryoglobulinaemia (MC) react, in 95% of cases, with MoAbs against the cross-reactive idiotypes (CRI) Cc1 or Lc1 (corresponding to the products of the VH1 and VH4 genes). MC is closely associated with HCV infection, a virus which infects lymphocytes and may replicate in B cells. It has been suggested that HCV may induce clonal selection of B cells producing monoclonal IgMk RF in type II MC. To verify whether HCV is enriched in B cells, and in the subsets expressing Cc1 and Lc1 CRI, we studied peripheral blood lymphocytes from eight patients with MC and HCV RNA-positive sera. Seven patients had RF reacting with anti-Cc1, the other with anti-Lc1 CRI. Total lymphocytes, T cells, B cells, and Cc1+ or Lc1+, Cc1- or Lc1- B cells were purified using MoAb-coated magnetic beads. Lymphocyte subsets were then diluted to give a range of 1 x 106-1 x 103 cells and tested for HCV RNA by reverse transcriptase-polymerase chain reaction. HCV was found exclusively in B cells in seven out of eight patients. In three patients HCV was enriched in the Cc1+ cells. In one of these patients, HCV was found exclusively in Cc1+ cells, with Cc1- cells being HCV-. The data indicate that B cells from type II MC patients are almost constantly infected by HCV. In selected cases, B cell subsets expressing IgMk RF CRI are the prevalent cell type infected by HCV. Our data suggest HCV involvement in B cell dysregulation leading to cryoprecipitable IgMk RF production.

Hepatitis C virus genotypes in Estonia.

Distribution of hepatitis C virus (HCV) geno(sub)types among 215 Estonian patients hospitalized with acute or chronic hepatitis and with HCV RNA-positive sera was investigated. For genotyping, both multiplex PCR with subtype-specific primers of the core region and RFLP analysis of cDNA of the 5' NCR region were used. These two methods permitted a correct characterization of genotypes, a more truthful characterization of mixed infections, and combined use of single-tube performances. They revealed, respectively, 200 and 202 (93.0% and 93.9%) HCV-positive samples of sera, subtype 1a- 0.9% and 0.9%, 1b- 56.3% and 64.2%, 3a- 13.9% and 22.3%, 2a- 6.5% and 5.6%, type 4 0.5% and 0%, mixed infections- 13.5% and 0%, and unidentified- 1.4% and 0.9%. In the majority of cases (84.7%) both methods gave completely or partially concordant results; in mixed infections, as determined by subtype-specific PCR, only one subtype was revealed by the RFLP method. In the remaining 15.3% of the cases (Ohno- 7.0%, RFLP- 8.3%) only one of the methods was positive. The epidemiological analysis of the dynamics of the subtypes' relative participation may indicate increasing 3a and decreasing 1b subtype infection during recent years.

Detection of antibodies to hepatitis C virus in seronegative patients using an immune complex dissociation assay.

Sera from a small percentage of hepatitis C virus (HCV)-infected blood donors do not react in the currently available assays for detection of antibody to HCV (anti-HCV) and, as a consequence, hepatitis C may develop in recipients of this blood. One possible explanation for this phenomenon is that antibody is present but cannot be detected because it is sequestered in circulating immune complexes. To test this hypothesis, an immune complex dissociation (ICD) assay was developed to disrupt any immune complexes that might be present in these anti-HCV-negative, HCV RNA-positive sera. A positive result in this test would indicate that antibody is present in these patients but is not detectable under routine anti-HCV testing conditions. Nine chronic and two acute HCV patients, all negative for antibody but positive for HCV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) were tested, together with appropriate controls. Three of the nine study patients with chronic HCV had evidence of anti-HCV after immune complex dissociation compared with none of the two patients with acute HCV. Although the number of patients tested was small, the negative results in the patients with acute HCV presumably indicates that anti-HCV seroconversion had not yet occurred. Incorporation of an ICD step into existing anti-HCV assays may enable blood banks to detect those rare instances of patients with chronic HCV who are antibody negative; this would minimize potential cases of post-transfusion hepatitis in recipients.

Detection of hepatitis C virus specific core protein in serum of patients by a sensitive fluorescence enzyme immunoassay (FEIA)

A protein-capture fluorescence enzyme immunoassay (FEIA) was developed using monoclonal antibodies (mAbs) against recombinant hepatitis C virus (HCV) core protein. Four hybridoma cell lines (5E3, 5F11, 515S, 1080S) were established and characterized. These monoclonal antibodies (mAbs) each had IgG1 and IgG2 isotypes, and recognized major B cell epitopes within the immunodominant nucleoprotein amino terminal subregion. Using mAb 5F11 as the first antibody to the solid phase and β- d-galactosidase-conjugated mAb 5E3 as the second antibody to the protein, we established a specific HCV core protein capturing FEIA capable of detecting as little as 20 pg/ml of recombinant HCV core protein. HCV core protein in serum was detectable after treatment with 4.0% polyethyleneglycol, 0.5 M NaOH, and 5% Triton X-100. The results of a peptide inhibition assay indicated that this FEIA is specific for HCV RNA positive sera. The quantity of HCV core protein detected in serum was significantly correlated to the level of HCV RNA. The detection limit for HCV core proteins was an HCV RNA per titer of ∼ 10 4/ml. Using this FEIA system, the detection ratio of HCV core protein in patients with chronic HCV infection was 92.3% ( 70 76 ).

Hepatitis C virus viremia in SCID-->BNX mouse chimera.

Chimpanzees are currently the only nonhuman animal model for reproducible propagation of hepatitis C virus (HCV). A chimeric mouse model was used for the induction of hepatitis C viremia, using BNX (beige/nude/X-linked immunodeficient) mice preconditioned by total body irradiation and reconstituted with SCID mouse bone marrow cells. HCV-infected liver fragments from patients with HCV RNA-positive sera were transplanted under the kidney capsule of the chimeric mice. HCV-specific RNA sequences were detected by reverse transcriptase nested polymerase chain reaction (RT-PCR) in serum of approximately 50% of grafted animals. In addition, normal liver specimens were incubated with HCV serum and transplanted into chimeric mice, leading to viremia in approximately 25% of animals. Sequential histologic evaluation of the liver implants, from day 2 to week 14 after transplantation, revealed loss of lobular architecture within the implants. However, viremia persisted for 10-50 days after transplantation. These results offer a new HCV model.