Adult ; Female ; Genotype ; Hepacivirus ; classification ; genetics ; Humans ; Male ; Turkey

OBJECTIVEIn 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.

METHODSThe 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.

CONCLUSIONThis 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.

5' Untranslated Regions ; chemistry ; DNA Restriction Enzymes ; Genotype ; Hepacivirus ; classification ; genetics ; RNA, Viral ; analysis

OBJECTIVETo determine the distribution of HCV genotypes among volunteer blood donors in Guangzhou.

METHODSSix-nine HCV RNA-positive samples were collected from volunteer blood donors in Guangzhou. NS5B fragments of HCV were amplified followed by DNA sequencing and phylogenetic analysis.

RESULTSHCV genotypes were determined for 67 samples. Among them, the subtypes 1b, 2a, 3a, 3b, 6a and 6n were detected at the frequencies of 37.31%, 4.48%, 7.46%, 4.48%, 44.78% and 1.49%, respectively.

CONCLUSIONHCV 1b and 6a are the most predominant two subtypes among volunteer blood donors in Guangzhou.

Blood Donors ; China ; Genotype ; Hepacivirus ; classification ; genetics ; isolation & purification ; Humans ; Phylogeny ; RNA, Viral ; genetics ; Sequence Analysis, DNA

OBJECTIVETo develop a new DNA chip with coloration, which can be used for rapid and economical detection of the genotyping of hepatitis C virus (HCV).

METHODSProbes and primers were designed according to the sequence of HCV 5' non-coding region (5' NCR) to fabricate DNA chip. Experimental group consisted of 60 positive serum samples and control group consisted of 20 negative serum samples. To obtain the aimed gene, then they were hybridized with DNA chip. Finally, the results showed in a nylon film. The results of DNA sequencing of samples were used as the control in double blind experimental.

RESULTSUsing DNA chip, HCV was detected in positive of all serum specimens of experimental group and negative in control group. The determination of HCV genotype by DNA chip showed corresponding rate of 96.7% with those by sequence assay.

CONCLUSIONIt showed higher specialty and sensitivity using DNA chip to detect the genotype of HCV. It would be valuable for the clinical genotyping of HCV

5' Untranslated Regions ; genetics ; Base Sequence ; Genotype ; Hepacivirus ; classification ; genetics ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Sequence Analysis, DNA

OBJECTIVEUsing polymerase chain reaction-reverse blot dot (PCR-RDB) technique to establish a new method for hepatitis C virus (HCV) genotyping and to study the distribution of HCV genotypes in Foshan area.

METHODSHCV primers and probes were designed in 5'-untranslated region (nt-1-nt-299) of HCV. HCV RNA in serum was isolated and purified, and its cDNA was obtained by reversed transcription. Nested PCR using biotin-labelled primers, was done. PCR products were hybridized with immobilized specific probes (genotype 1a to 3b) on Biodyne C membrane to genotype HCV by color development while adding POD and TMB. A certain judgment could be made according to the position of color reaction. The reliability of this new method was verified by sequencing. HCV RNA levels in serum were determined by real time fluorescent quantitative (FQ)-PCR. 60 FQ-PCR-positive HCV sera from Foshan area were genotyped using this assay.

RESULTSAll 60 sera could be successfully genotyped by PCR-RBD. 50 (83.3%) cases were found to be genotype 1b, 2 (3.3%) as genotype 1a and 2 (3.3%) as genotype 2a while 5 (8.0%) to be mixture of genotype 1a and 1b, and 1 (1.7%) to be mixture of genotypes 1b and 2a. No genotypes 2b, 3a and 3b were found. The results of PCR-RDB genotyping methods coincided with sequence analysis.

CONCLUSIONNewly established HCV genotyping system was proved to be sensitive, specific, precise and economic, thus suitable for clinical and epidemiologic studies. The results of HCV genotyping showed that genotype 1b was the predominant genotype in Foshan area.

Genotype ; Hepacivirus ; classification ; genetics ; Hepatitis C ; virology ; Humans ; Immunoblotting ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

BACKGROUNDNumerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed.

METHODSBased on 45 sequences, representing HCV genotypes 1 - 6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182 - 2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1 - 6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis.

RESULTSThe correspondence of amino acids in HCV NS5A region with amino acids in positions 2182 - 2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182 - 2211 (R1), 2272 - 2301 (R7) and 2302 - 2331 (R9): the highly variable 2212 - 2241 (R3) and 2257 - 2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis.

CONCLUSIONSThe major antigenic regions in HCV NS5A region were at 2212 - 2241 (R3), 2272 - 2301 (R7) and 2302 - 2331 (R9). Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

Amino Acid Sequence ; Genotype ; Hepacivirus ; classification ; genetics ; Humans ; Molecular Sequence Data ; Serotyping ; Viral Nonstructural Proteins ; chemistry ; immunology

Alanine Transaminase ; blood ; Genotype ; Hepacivirus ; classification ; genetics ; Hepatitis C, Chronic ; drug therapy ; immunology ; virology ; Humans ; RNA, Viral ; blood

Hepacivirus ; classification ; genetics ; Hepatitis C ; diagnosis ; Hepatitis C Antibodies ; blood ; Humans ; RNA, Viral ; blood ; Viral Core Proteins ; blood

To investigate risk factors for HCV infection according to the genotype, we stud-ied 178 patients positive for HCV-PCR and 226 controls that were negative for the anti-HCV antibody. One hundred and twenty five controls (community con-trol) were recruited from spouses of HCV-PCR-positive patients and the other 101 from hospital visitors (hospital control). HCV genotyping was performed by PCR, and epidemiological data were obtained from all participants. The distribu-tion of HCV genotypes was as follows - 1a (0.6%), 1b (39.9%), 2a (38.2%), 2b (0%), 3 (1.1%), and unclassified (20.2%). By multivariate analysis, blood transfu-sion (OR 2.90) and endoscopy (OR 2.80) were found to be risk factors for HCV genotype 1b versus the community control. Similarly, blood transfusion (OR 3.17) was found to be risk factors for HCV genotype 1b versus the hospital control. Blood transfusion (OR 2.75) and endoscopy (OR 3.57) were risk factors for HCV genotype 2a versus the community control, and blood transfusion (OR 4.55) and endoscopy (OR 2.16) were those versus the hospital control. Our results suggest that the risk factors for HCV infection are similar among the different genotypes. Blood transfusion and endoscopy were found to be associated with HCV infection.
Female ; Genotype ; Hepacivirus/classification/genetics ; Hepatitis C/*virology ; Humans ; Korea ; Male ; Middle Aged ; Multivariate Analysis ; Risk Factors

OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe HCV genotype distribution in northern and southern regions were similar, but was significantly different between the patients infected through transfusion and other routes.

5' Untranslated Regions ; Adolescent ; Adult ; Aged ; China ; Female ; Genotype ; Hepacivirus ; classification ; genetics ; isolation & purification ; Hepatitis C, Chronic ; etiology ; genetics ; transmission ; Humans ; Male ; Middle Aged ; Transfusion Reaction