| ObjectivesRecent data from"China information system for disease control and prevention"show that the number of reported cases of hepatitis C has been increased year by year. The fact of that people infected by Hepatitis C shown minimal symptoms at the very beginning make it easily developed into chronic hepatitis, cirrhosis and hepatocellular carcinoma, that hepatitis C has been become a major public health problem. Research on epidemiologist and pathogenic of Hepatitis C was less in China. There are no large-scale epidemiological studies conducted since a survey on HCV antibody positive-rate in natural population had been carried out in 1992 in Fujian province, and there are no articles about the genotyping of HCV in Fujian province have been reported. In addition, different genotypes of HCV with different sensitivity to the antiviral, and researches on pathogenesis, vaccine and drug screening of HCV have been hampered by absence of in vitro model in a long-term. Therefore, in order to prevent and control hepatitis C better, it is necessary to analyze the prevalence characteristics of hepatitis C in Fujian province, obtain basic information about epidemic situation, establish prediction model of HCV reported cases, and provided evidence for prevention strategy making. Meanwhile, construction of an in vitro cell culture system of the strain prevalence in Fujian province can provide a platform for basic research of HCV. Drug screening carried out in this platform can be a practical guidance to clinical administration of medicines.Methods1.A cross-sectional research, by multi-stage sampling method, was conducted to obtain the basic information of hepatitis C epidemic situation; ELISA test was used to detected hepatitis C virus antibody in serum, and to get positive rate of anti-HCV among natural populations in Fujian Province in 2006;2. The serum samples of voluntary blood donors, drug addicts and patients with anti-HCV positive were collected. RT-PCR method was used to amplify the sequence in C/E1 and NS5B region respectively, sequences were analyzed and compared by biological software after cloning and sequencing, and phylogenetic trees were constructed to analyze features of gene distribution among different groups of people in Fujian Province ;3. According to data of monthly incidence of HCV reported in"China information system for disease prevention and control"from 2004 to 2010 in Fujian Province, we establish the fittest disease prediction model by analyzing data characteristics.4. Full-length cDNA in different subtypes was obtained by RT-PCR by assembling 3 fragments to constructed a recombinant plasmid; the chimeric constructed by a nearly full-length genome of Fujian strains and non-structural areas of JFH1 strain was transcript in vitro and was transfected into Huh7 cells to develop a in vitro cell culture system in different subtypes. It was identified by IFA, RT-PCR and other methods, and was initial used in vitro drug screening.Results1. The anti-HCV positive rate of natural population Fujian province was 0.7%. There was no difference in regional distribution, and no difference between men and women. Anti-HCV positive rate was increasing with age growth, especially in the age group above 50 years, and the anti-HCV-positive rate is far higher than age group below 50 years. In combination with other data, that the anti-HCV positive rate in natural populations in this survey shows a downward trend compared with positive rate in 1992, although the later one had been found having high false positive rate, and current situation of HCV is not optimistic.2. The major epidemic genotype of hepatitis C in Fujian Province are 6a and 1b, followed by 3b and 2a, the genotype prevalence between different groups differ from each other. The prevalent genotype in patients is 1b, and in voluntary blood donors and drug addicts as the same of 6a, differences between patients and other group may indicate the changing of major epidemic genotype from 1b to 6a.And mixed infections was also been observed, that mainly showed in type 3b reorganization with other subtypes3. Disease surveillance data from"China Information System for Disease Prevention and Control " show that since 2004, after the implementation of network report, the number of reported hepatitis C cases showed an increasing trend, and it is mainly caused by the rising in the number of reported clinically diagnosed cases, while the number of laboratory-confirmed cases remained stable. The rising in the number of clinically diagnosed cases may have been caused by reasons below: increased awareness of reporting, development of infected carrier to patients, inconsistency of diagnostic criteria.4. ARIMA (2,1,0) (2,1,0) 12 (without constant) model do well in tracking and prediction the monthly reported cases of hepatitis C, but due to the impact of external factors changes, it is better in short-term forecasts than long-term forecast.5. Amplified and assembling six strains of full-length genes of hepatitis C virus in diffrent subtypes (including two strains of subtype 1b, and four strains of subtype 3a, 3b, 6a and 6n each) and a half-length fragment of subtype 1a; constructed the whole length recombinant plasmid pBS1, pBR2, pBSR5, pXX4, pXX19 and pXQ124.6. Successfully constructed the pBS1/JFH1 (6a/2a) chimeric in vitro cell culture system. Linearized plasmid pBS1/JFH1 produced infectious RNA by in vitro transcription, the infectious RNA was transfected to Huh7 cells, and the derived virus can cause infection of the new Huh7 cells. The infectivity of chimeric is relatively weaker than pJFH1strain.7. Preliminary use pBS1/JFH1 (6a/2a) chimeric in vitro cell culture system in drug screening. The results show that the INFαcan effectively inhibit viral replication with dose dependent. RBV, NTZ, and CsA also could inhibit HCV viral replication alone, but requires a higher dose. And combination using of these three drugs with the INFαcan significantly improve the antiviral effect of INFαin HCV. Conclusions1. Anti-HCV positive rate of natural population in Fujian province show a downward trend, but reported incidence in surveillance increased year by year at present. It is due to the characteristics of chronic tendency of HCV, the higher incidence today is the result of the higher positive rate of HCV years before.2. Analyzed the domain genotype of HCV in different groups of people in Fujian Province, and found that the domain genotype of HCV may have changed from 1b to 6a and mixed infections exist in part of the infectious carrier in current.3. Amplified and assembled six strains full-length gene of HCV in five subtypes, and successfully constructed pBS1/JFH1 (6a/2a) chimeric in vitro cell culture system. It is feasible for this system to be used in drug screening, and it is enabling tool for further basic research, such as to explore the virulence gene, pathogenic mechanisms and so on.
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