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 establish hepatitis C virus (HCV) infected cell model which is similar to the infection in vivo and can support HCV to replicate for a long time.

METHODSAfter infected with HCV-positive serum, Hep G2 cells were cultured for 60 days. Nested RT-PCR was used to detect plus and minus HCV RNA in cultured cells and supernatants.

RESULTSPlus HCV RNA was detected intermittently in Hep G2 cells during 2-30 days, minus HCV RNA was detected during 3-30 days after infection, the detection rate was similar to plus HCV RNA. Plus and minus HCV RNA can be still intermittently detected during 31-60 days after infection. However, the detection rate gradually declined. Plus HCV RNA was also found intermittently positive in the supernatant, and the detection rate was consistent to that in cells. Minus HCV RNA was not detected in the supernatant.

CONCLUSIONHep G2 cells were susceptible to HCV, and could support HCV to replicate for a relatively long time. Hep G2 is an ideal HCV infection cell model.

Cell Line, Tumor ; Hepacivirus ; genetics ; growth & development ; Humans ; RNA, Viral ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication

OBJECTIVETo study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5'UTR.

METHODSPlasmids, 5'UTR-Luc(+) and 5'UTR-Luc(-) carrying the forward and reverse DNA sequences corresponding to the HCV 5'UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5'UTR sequence was cloned into a GFP vector to make 5'UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy.

RESULTS5'UTR-Luc(+) had an obvious luciferase activity whereas 5'UTR-Luc(-) had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the cells transfected with the plasmid of 5'UTR-EGFP.

CONCLUSIONThe forward DNA sequence corresponding to HCV 5'-UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.

5' Untranslated Regions ; genetics ; Carcinoma, Hepatocellular ; virology ; DNA, Viral ; genetics ; Hepacivirus ; genetics ; isolation & purification ; Humans ; Liver Neoplasms ; virology ; Luciferases ; metabolism ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

OBJECTIVETo screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system.

METHODSHepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank.

RESULTSAfter BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC.

CONCLUSIONT7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Cell Line ; Gene Library ; Hepacivirus ; metabolism ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; Viral Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

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

The mortality due to chronic liver disease, including liver cirrhosis and hepatocellular carcinoma (HCC), ranks as one of the highest in Korea. The prevalence rates of hepatitis C virus (HCV) and hepatitis B virus (HBV) infections in the general Korean population are approximately 1 and 5%, respectively. Blood transfusion was the strongest risk factor for the transmission of HCV infection. Therefore, the evaluation of risk factors for HCV infection including blood transfusion, intravenous drug user, hemophilia, and hemodialysis, is important. The most prevalent HCV genotype is 1b followed by 2a. The annual incidence of HCC among HCV-related liver cirrhosis has been estimated at 5%, and approximately 12% of HCC is attributable to HCV and 68% to HBV in Korea. HCV infection is more closely associated with HCC in elderly patients than HBV-related HCC. Even though the prevalence of anti-HCV in Korea has been reduced and the risk of HCV transmission through blood transfusion has markedly decreased, public-health programs to prevent de novo infections should be developed. This review describes the HCV prevalence and risk factors among the general population, and the distribution of HCV genotypes as well as the clinical course of HCV in Korea.
Adult ; Carcinoma, Hepatocellular/*virology ; Genotype ; Hepacivirus/genetics/isolation & purification ; Hepatitis C, Chronic/*complications/*epidemiology ; Humans ; Korea ; Liver Cirrhosis/*virology ; Liver Neoplasms/*virology ; Middle Aged ; Prevalence ; Risk Factors

Blood donor recruitment models have changed from paid donors to employer-organized donors and to voluntary donors in China. Reports on the hepatitis C virus (HCV) infection among voluntary blood donors in China have been rarely found at present. The prevalence of anti-HCV and genotypes among the first-time voluntary blood donors was investigated in Chongqing area of China. A total of 13,620 serum samples were collected from the first-time voluntary blood donors in Chongqing, China. Anti-HCV antibody was tested by ELISA. The Core/E2 region of HCV RNA from HCV seropositive samples was amplified by RT-PCR for genotyping. The results indicated that the prevalence of anti-HCV averaged 0.49% (67/13,620), and the highest rate (0.86%) was obtained in the group aged 40 to 49. A higher prevalence was observed among the more educated donors, and metropolitan donors. The ratios of following genotypes 1b, 2a, 3a and 3b were 4 (18%), 5 (23%), 9 (41%) and 4 (18%) in all the 22 samples respectively. Genotype 3 (3a and 3b) was the predominant genotype. In conclusion, the prevalence of anti-HCV was low among the population of voluntary blood donors in Chonqing area. The genotyping results showed the possibility of presence of druggies among the voluntary blood donors. Therefore, more attention should be paid to exclude those high-risk persons from the volunteers.
Blood Donors ; China ; epidemiology ; Genotype ; Hepacivirus ; genetics ; isolation & purification ; Hepatitis C ; epidemiology ; transmission ; Hepatitis C Antibodies ; blood ; Humans ; Incidence ; Seroepidemiologic Studies

OBJECTIVETo survey the application of PCR for screening HCV RNA from blood donations within the window period.

METHODSAccording to a standardized method, 12 blood banks organized by the National Center for Clinical Laboratories collected and prepared about ten thousands specimens. The specimens were tested with two different kits.

RESULTSAmong the 7173 specimens A group, 21 were PCR positive for HCV RNA. The positive rate was 0.29%. There were not positive for HCV RNA among 7477 specimens (B group).

CONCLUSIONSIt is feasible to use the PCR screening for the detection of HCV RNA of blood donations but is unnecessary to standardize the specimen collection and the kit selection.

Blood Donors ; Blood Transfusion ; Hepacivirus ; genetics ; isolation & purification ; Hepatitis C ; prevention & control ; Humans ; Mass Screening ; Polymerase Chain Reaction ; RNA, Viral ; analysis

OBJECTIVETo investigate the distribution of hepatitis C virus (HCV) genotypes in Yixing, Jiangsu province.

METHODSGenotypes identification on sera samples were obtained from 158 donors who had already been anti-HCV positive through PCR method with type specific primer designed according to the sequence of 5'non-coding region (5'NCR). 5'NCR was also sequenced and compared with published date. Genotypes distribution was investigated in patients with different sex and clinical types of hepatitis C.

RESULTSOf the total 158 patients, 95 were HCV RNA positive in which 80 patients having genotype 1b (80/95; 84.4%), 5 patients having genotype 2(5/95; 5.3%), 5 patients with 1b/2 mixed genotypes (5/ 95; 5.3%) and another 5 patients whose genotype undetermined. The difference on the distribution of HCV genotypes was significant between female and male patients (P < 0.05) but not in different kinds of hepatitis C patients.

CONCLUSIONType 1b was the predominant HCV genotype in Yixing area.

Base Sequence ; Blood Donors ; China ; epidemiology ; Female ; Genotype ; Hepacivirus ; genetics ; isolation & purification ; Hepatitis C ; epidemiology ; prevention & control ; therapy ; virology ; Humans ; Male ; Middle Aged ; Sequence Analysis, DNA ; Sex Factors