Evaluation of the performance of the EIAgen HCV test for detection of hepatitis C virus infection.

The EIAgen HCV test (Adaltis Inc., Montreal, Canada) is an enzyme immunoassay (EIA) for the detection of anti-hepatitis C virus (HCV) antibodies. This study compared the performance of this test side-by-side with the current Ortho HCV 3.0 Anti-HCV assay (Ortho-Clinical Diagnostics Inc., Johnson & Johnson Company, Raritan, NY, USA). Among 2559 specimens examined, 178 were true positives, 2376 were true negatives and 5 were indeterminate. The sensitivity of the EIAgen HCV test was 100%, versus 98.3% for the Ortho HCV test, while their respective specificities were 98.1% and 98.2%. The EIAgen HCV test gave a positive predictive value of 79.8% and a negative predictive value of 100%. Overall, the concordance of this test with the Ortho HCV test was 98.2%. Specimens from potentially interfering substances, such as sera from pregnant women, sera from patients with acute non-C hepatitis, autoimmune diseases, lipidemia, or from patients undergoing hemolysis, showed no interference with either EIA. An EIAgen HCV test signal-to-cut-off ratio of >5.9 would be highly predictive of a true-positive finding in these specimens. The EIAgen HCV test is well suited for screening blood and blood products in antibodies to HCV.

[Hepatitis B surface antigen terminates codon bias selection].

OBJECTIVE: To investigate the existence condition of hepatitis B surface antigen(HBsAg) termination codon bias. METHODS: A total of 174 reference sequences of all kinds of Hepatitis B virus(HBV) genotypes were chosen from GenBank, and compared by BioEdit. Then secondary structure of RNA was constructed and analyzed together. RESULTS: (1) There were two types of HBsAg termination codon: TAA and TGA in 174 reference sequences. TAA was in 124 cases (71.26%); and TGA in 50 cases (28.74%). (2) There was codon bias selection in HBsAg termination codon, and it could affect the secondary structure of RNA and amino acid sequence encoding protein. CONCLUSION: HBsAg termination codon bias exists and may be related to RNA structure and the conservation of protein function in the evolutionary progress.

Senescence marker protein 30 in acute liver failure: validation of a mass spectrometry proteomics assay.

BACKGROUND: Our previous proteomic study showed that the senescence marker protein (SMP30) is selectively present in the plasma of a murine model of acute liver failure (ALF). The aim of this study was to validate this SMP30 expression in the plasma and liver tissues of mice and humans with ALF. METHODS: After the proteomic analysis of plasma from a murine model of D-galactosamine/lipopolysaccharide (GalN/LPS)-induced ALF by two-dimensional electrophoresis (2-DE) and mass spectrometry, the expression levels of SMP30 in the plasma and liver tissues were validated by western blot and RT-PCR analyses. These results were then confirmed in plasma samples from humans. RESULTS: These data validate the results of 2-DE, and western blot showed that SMP30 protein levels were only elevated in the plasma of ALF mice. Further analysis revealed that GalN/LPS induced the downregulation of SMP30 protein levels in liver tissues (by approximately 25% and 16% in the GalN/LPS-treated mice and in the treated mice that survived, respectively; P < 0.01). Hepatic SMP30 mRNA levels decreased by about 90% only in the mice that survived the GalN/LPS treatment. Importantly, plasma obtained from patients with ALF also contained higher levels of SMP30, about (3.65 +/- 0.34) times those observed in healthy volunteers. CONCLUSION: This study shows that SMP30 is not only a potential biomarker for the diagnosis and even prognosis of ALF. It also plays a very important role in a self-protective mechanism in survival and participates in the pathophysiological processes of ALF.

The functional evaluation of dendritic cell vaccines based on different hepatitis C virus nonstructural genes.

Hepatitis C virus (HCV) nonstructural (NS) genes are relatively conserved and play critical roles in cellular immune responses against HCV. The aim of the study was to evaluate the immunogenicity of the different HCV NS genes through transduction of DCs and presentation to T cells. Monocyte-derived DCs from healthy donors were infected with the recombinant adenovirus (Ad) harboring HCV NS3 (AdNS3), NS4 (NS4A and NS4B; AdNS4), NS5 (NS5A and NS5B; AdNS5), NS3/NS4 (AdNS3/NS4), and NS4/NS5 (AdNS4/NS5) genes, and then used to stimulate autologous lymphocytes in vitro. Antigen-specific cellular immune responses were detected by interferon-gamma (IFN-gamma), interleukin 4 (IL-4), and Granzyme B (GrB) enzyme-linked immunospot assays (ELISPOT). DCs expressing different HCV NS genes all induced positive immune responses. Furthermore, DCs transfected with AdNS3/NS4 were superior to DCs infected with AdNS3 or AdNS4 in inducing HCV-specific immunity. The same results were obtained when we compared DCs infected with AdNS4/NS5 to AdNS4 or AdNS5. DCs transduced with NS3/NS4 or NS4/NS5 had similar ability to elicit specific immune responses to HCV.

[The distribution of hepatitis C virus genotype 1a in Yanbian area].

OBJECTIVE: To investigate the distribution of Hepatitis C Virus (HCV)genotype 1a in Yanbian area, Jilin province. METHODS: The genotypes of the serum specimens of 47 patients with HCV from Yanbian area were determined by Scheme ABC of RFLP based on HCV 5' noncoding region (5'NCR). The samples of type 1a (Y2, Y4, Y6, and Y8) were amplified from the 5'NCR and NS5B regions by RT-PCR, and then sequenced directly. These sequences were subjected to phylogenetic analysis with 27 reference sequences of HCV complete genomes from GenBank. RESULTS: 44 specimens were HCV RNA positive, among which 19 specimens (43.2%) were of the type 1a/1b, 12 (27.3%) were of the type 1b, 8 (18.2%) were of the type 2a/1b, 4 (9.1%) were of the type 1a, and 1 (2.3%) was of the type 2a, however, the genotypes 2b and 3 approximately 6 lacked. The phylogenetic analysis for 1a samples showed that according to 5'NCR they belonged to type 1a, while on NS5B belonging to type 1b. The most nucleotide identity of 5'NCR was respectively 0.990, 0.990, 0.990, and 0.990 between Y2, Y4, Y6, Y8 and the isolate HC-J1 (genotype 1a), while that of NS5B was respectively 0.936, 0.957, 0.936, and 0.936 between Y2, Y4, Y6, Y8 and the isolate HC-J4 (genotype 1b). CONCLUSION: In Yanbian area the distribution of HCV genotype is different from those in other areas and 1a/1b has even replaced 1b as the most HCV genotype here. The results of genotyping 1a on 5'NCR and NS5B are completely inconsistent. This phenomenon may be the consequence of recombination in evolution.

[Utilization of Uracil-DNA glycosylase for combining reverse transcription and anti-contamination with polymerase chain reaction in hepatitis C virus].

OBJECTIVE: To develop a hepatitis C virus(HCV) reverse transcription-polymerase chain reaction (RT-PCR) assay using Uracil-DNA glycosylase (UDG) for amplicon contamination control and evaluate the temperature and UDG concentrations for anti-contamination. METHODS: In this new HCV RT-PCR assay, reverse transcription, UDG anti-contamination and the first PCR were carried out at the same time. The layer candles were used to prevent the contamination in the second PCR. dU-DNA was used as quality control for UDG anti-contamination and templates to determine the sensitivity of the new HCV RT-PCR assay. HCV cDNA was detected by DNA enzyme immunoassay (DNA-EIA). RESULTS: Complete degradation of amplicon DNA was observed on the conditions of 0.2au UDG per reaction volume respectively at 37 degrees C and 42 degrees C for 40 min. The anti-contamination condition also could eliminate all detectible dU-DNA, including the highest concentration of amplicon DNA.The 1:10(4) dilution of the HCV RNA sample containing 2.110x 10(5)copies /mL copies of RNA could be detected. CONCLUSION: Our results indicate that this new RT-PCR assay can control the contamination stringently and is sensitive as well.

[Concordance between hepatitis C virus serotype and genotype in chronic hepatitis C patients in China].

OBJECTIVE: To investigate the relationship of hepatitis C virus (HCV) serotype with genotype. METHODS: The serotypes of HCV in the serum of 104 patients with chronic hepatitis C from 14 cities in China for which HCV genotypes were available, were determined by ELISA using Murex HCV Serotyping 1-6 Assay. RESULTS: The serotypes of 86 (82.69 percent) of the 104 serum specimens were determined, and HCV serotypes were determined for 91 strains. Overall the concordance between hepatitis C virus serotype and genotype was 62.1 percent, and the concordance of serotype, with genotypes 1, 2 and 3 were 69.4 percent, 51.2 percent and 70.0 percent, respectively. The false-negative rate and concordance of genotype 2b was lower (54.5 percent). CONCLUSION: The specificity of HCV serotyping was affected by HCV strains' genotype and sometimes HCV serotype was not in concordance with genotype.

[Analysis and state of HCV genotype 6a infection].

OBJECTIVE: To investigate the infection state of hepatitis C virus genotype 6a in China. METHODS: Three (95, 126, 150)HCV genotype 6a serum samples were identified by digesting 5'NCR with compound enzyme method. Then, HCV 5'NCR and NS5B fragments were amplified from these samples by RT-PCR assay and sequenced. The phylogenetic trees of the samples were analyzed and compared with 24 HCV complete gene sequences from GenBank. RESULTS: The sequencing reports on 5'NCR showed "CA" bases in 3 serum samples (95,126,150) were inserted into -145 site, and the sequences of 3 serum samples had the highest homology with sequence Y12083( 0.934, 0.930, and 0.926, respectively). The results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 6a. The sequencing reports on NS5B showed the 3 serum samples also had the highest homology with HC-J4(0.934, 0.930, and 0.926, respectively), and the results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 1b. To exclude the influence of amplification efficiency of primers, NS5B fragments were amplified by HCV genotype 6a specific primers and no amplification products appeared. CONCLUSION: There are different results of HCV genotype by analyzing 5'NCR and NS5B in 3 samples infected with HCV genotype 6a. It may be related with gene recombination. It suggests HCV genotype should be analyzed on more than two regions.

[Hepatitis c virus genotype research by ABC programs of 5'-NCR restriction endonuclease digestion].

OBJECTIVE: In order to fully understand hepatitis c virus (HCV) genotype 3b, 1a, 2b and 6a infection in China, We built HCV 5'-noncoding region (5'-NCR) of different genotypes and subtypes. METHODS: The classification HCV into variable genotypes (subtypes) was carried on by programs A, B and C A. Using a combination of three restriction endonuclease BHH' (BsrB I, Hae II, Hinf I) digestions at the same time. The distinct genotypes were classified into 5 groups: genotype 1 (1a, 1b), 6a, 2 (2a, 2b), genotype 3 (3a, 3b), genotype4 (4a). B. With regard to genotype 1, we could distinguish subtype 1a from 1b using BstU I digestion. C. Using restriction endonuclease Hae III, genotype 2a, 2b, 3b, 4a, 6a are differentiated respectively. RESULTS: (1) HCV genotype 1a, 1b, 2a, 2b, 3a, 3b, 4a, 6a are fully discriminated by comparison with the genotypes regular samples. (2) Of the 93 patients, HCV genotype distribution in China was 66.67% for 1b, 18.28% for 2a, 3.23% for 1b/2b, 3b, 2b respectively. 2.15% for 2a/2b, 1b/2a respectively. 1.08% for 1a. CONCLUSION: This research indicated that adoption of HCV 5'-NCR A B C restriction endonuclease digestions techniques, might be sensitive and efficient to detect HCV and discriminate HCV genotype (subtypes) 1a to 6a.

[The difference in distribution of HCV genotypes between patients infected with HCV by transfusion and non-transfusion routes].

OBJECTIVE: To investigate the HCV genotypes distribution in northern and southern cities in China and the difference between patients infected with HCV by transfusion and non-transfusion routes. METHODS: The HCV genotypes of the patients with chronic hepatitis C from 9 cities belonging to different regions were genotyped by the PCR products of 5 prime untranslated region NTR digested with restriction endonucleases, and the HCV genotypes distribution among different cities or between the patients infected with HCV through transfusion and other routes was analyzed. RESULTS: The HCV genotypes of 214 in 219 cases were determined; 197 patients were infected with monogenotype HCV. The major epidemic genotypes of HCV isolates in China were 1b (76.64%) and 2a (18.22%), but 5.14% of patients were infected with HCV belonging to genotype 3b and this was the first report that there is genotype 4a in China. The HCV genotype distribution was not different in northern and southern areas, but was significantly different between patients infected with HCV through transfusion and non-transfusion routes (P=0.036). In patients infected trough transfusion, the rates of monogenotype HCV infection and genotype 1b were 93.88% and 76.87%, respectively, which were higher than those (86.57% and 58.21%) in the patients infected with HCV through non-transfusion routes. The rate of patient infected with mixed genotype HCV strains in non-transfusion group was 13.43%, which was higher than that (6.12%) of patients in transfusion group. CONCLUSION: The HCV genotype distribution in northern and southern regions were similar, but was significantly different between the patients infected through transfusion and other routes.