| Hepatitis C virus (HCV) is a major etiological agent of chronic hepatitis, cirrhosis and liver cancer. Currently, it is estimated that about 180 million people in the world infected with HCV (average rate of infection:3%). In China, HCV is second etiological agent causing chronic hepatitis, which is next to hepatitis B virus (HBV). According to the national epidemiological survey of viral hepatitis in 2002, the positive rate of anti hepatitis C antibody in our country was 3.2%, and estimated that there were more than 40 million cases with HCV infection. Approximately 50% to 80% of patients who have acute hepatitis C infection will develop into chronic hepatitis. Among these,20% to 30% will develop into progressive disease leading to the liver cirrhosis and hepatocellular carcinoma (HCC) after 20 to 30 years of infection. According to statistical data, over 50% liver transplantation arised from HCV related end-stage liver disease. And because HCV infection possesses chronic and hidden features, with the progression, the number of hepatitis C related end-stage liver disease patients will continue to go up in the next two decades, causing a huge burden of disease.HCV is belonging to Flaviviridae, and a member of hepatitis virus. Its genome is a single strand RNA, with whole genome about 9.6kb. Since RNA-dependent RNA polymerase lacks proofreading capability, the RNA genome of HCV was demonstrated great heterogeneity at the sequence level. HCV genetic diversity was found mainly at four levels:(1) The heterogeneity of HCV genome nucleotide sequence>30%, as the genotype, and there are six genotypes; (2) The heterogeneity> 25%, as the subtypes, there are currently more than 80 subtypes; (3) HCV genome nucleotide variation>10%, as isolates; (4) Quasispecies, exsiting in the patients' body, is a variety of different sequence composition, and has a high homology (homology≥95%) of HCV mutant groups. Of the four levels, research on HCV genotypes and subtypes has important epidemiological and clinical significances:(1) HCV genotypes exhibited different geographic and populational distribution. The distribution of HCV genotypes is also unstatic and the migration of HCV genotypes is continued to change from one area to another. Because of population migration and anti-HCV screening of blood donors, the HCV genotype distribution in China is gradually changing, especially in southwest provinces and southeast provinces. (2) Different genotypes have different clinical manifestations and severity of liver disease. Most scholars believe that the genotype 1 associated with the occurrence of liver cancer. Genotype 1, particularly subtype 1b, is probably progressed to severe liver diseases. Genotype 3 is a independent predictor for liver steatosis. (3) HCV genotypes are associated with the response to interferon (IFN) based therapy. Patients with genotypes 1 and 4 generally exhibit a poorer response to IFN-based therapy than those with genotypes 2 and 3. (4) Further research on HCV genotypes is important for development of the vaccine.In our study, firstly, HCV highly conserved region (5'UTR) and phylogenetically informative region (NS5B) were chosen to determine genotypes and subtypes by phylogenetic analysis, to clarify the characteristics of HCV genotypes and subtypes in Guangdong area. Then based on LiPA technology principle, we designed specific HCV primers and probes targeting the 5'UTR and Core region for subtypes 1b,2a,3a, 3b and 6a to develop a new HCV genotyping method with polymerase chain Reaction-reverse blot dot (RBD) technique. And finally, HCV subtypes distribution was investigated to understand the epidemiological and clinical significance in Guangdong area.The thesis has three parts as follow:1. HCV genotypes and subtypes determined by sequencing and phylogenetical analysis from both highly conserved region and informative region.HCV highly conserved region (5'UTR) and phylogenetically informative region (NS5B) were chosen to determine genotypes and subtypes from 99 chronic hepatitis C patients positive for both anti-HCV and HCV RNA. Direct nucleotide sequencing and phylogenetic analysis were carried out.99 cases were all accurately genotyped, of which all the cases were genotyped in 5'UTR, and 74 cases were determined in NS5B region as following:subtype 1a,1 case; subtype 1b,59 cases; genotype 1,7 cases; subtype 2a,11 cases; subtype 3a,2 cases; subtype 3b,2 cases; genotype 3,3 cases; subtype 6a,12 cases. There were 2 cases with different genotypes in the two regions, which were 6a/lb and 3a/lb respectively in 5'UTR/NS5B. There were 74 cases got positive results in both regions.72 cases were consistent,2 were inconsistent. Subtype lb in Guangdong area has three main isolates by phylogenetic analysis in NS5B region. This study demonstrated that phylogenetic analysis based on NS5B region sequences could accurately detect HCV subtypes and even the isolates within the same subtype but it was not sensitive. Phylogenetic analysis based on 5'UTR was highly sensitive but its ability to identify subtypes was insufficient. Combination with NS5B and 5'UTR sequence analysis could improve the accuracy and sensitivity of HCV genotyping. 2. Development and application of a new HCV genotyping method with polymerase chain reaction reverse dot blot technique.Based on the principle of LiPA technique, we designed the HCV primers and probes targeting the 5'UTR and Core region according to the sequences of 1b,2a,3a, 3b and 6a, and then developed a new HCV genotyping method with PCR reverse dot blot (RDB) technique.115 HCV RNA positive sera from chronic hepatitis C patients in NanFang Hospital were tested by this method; 111 sera were successfully genotyped and positive rate was 96.5%(111/115).15 negative control sera were all negative and the specificity was 100%. Among the 111 cases, subtypes were 1b 56.8%(63/111),2a 8.1%(9/111),3.6%(4/111),3b 5.4%(6/111) and 6a 25.2% (28/111) respectively.38 of these cases were verified by sequencing and phylogenetic analysis in NS5B region, the accuracy of RBD was 100%(38/38). The RBD genotyping method was confirmed to be sensitive, specific and economic, and is suitable for clinical and epidemiologic investigation in China.3. The clinical significance and epidemiological characteristics of HCV subtypes in Guangdong area.In this study, reverse dot blot method was used to detect HCV subtypes of chronic hepatitis C patients in NangFang hospital. Patients were followed up and the epidemiological and clinical significance of HCV subtypes distribution in Guangdong area were investigated.187 cases were detected and the distribution HCV subtypes was:subtype 1a,1.1%(2/187); subtype 1b,64.2%(120/187); subtype 2a,8.6% (16/187); subtype 3a,4.3%(8/187), subtype 3b,2.1%(4/187); subtype 6a 19.8% (37/187). The routes of transmission and HCV subtypes among different age groups were studied. It was found that blood transfusion was the main routes of transmission and subtype 1b was the main subtype in the group of older than 40y when compared with the group younger than 40yd (P<0.001). The ages and sex proportion were different between different genotypes. The patients with HCV subtype 1b were older and female dominant, and the patients with HCV subtype 6a were younger and male dominant. There was no significant difference between different genotypes in virus load, biochemical test, fasting blood glucose test and so on.Conclusions:1,HCV subtypes were accurately detected by phylogenetic analysis based on the NS5B region sequences, and HCV subtype lb with 3 main isolates was demonstrated in Guangdong area.2,Phylogenetic analysis based on 5'UTR had a high sensitivity but the ability to distinguish subtypes was insufficient.3,Combination sequence analysis with both 5'UTR and NS5B regions of HCV could improve the accuracy and sensitivity of genotyping.4,The PCR reverse dot blot method established in this study could detect HCV subtypes 1b,2a,3a,3b and 6a accurately. It would be suitable for clinical and epidemiologic investigation in China.5,HCV subtype 1b (64.2%) was still the main subtype prevalent in Guangdong area, followed by 6a (19.8%),2a (8.6%), and then 3a and 3b.6,The main route of transmission in the younger age group (<40 years) was non blood transfusion and the main subtype was non-1b.7,The ages and sex proportion were different between different genotypes. The patients with HCV subtype 1 b were older and female dominant, and the patients with HCV subtype 6a were younger and male dominant. There was no significant difference between different genotypes in virus load, biochemical test, fasting glucose test and so on.
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