| Background and Aims:Hepatitis C is an important infectious disease which threatens people's health. Approximately 17 million people are infected with the hepatitis C virus (HCV) worldwide. The overall prevalence of HCV in China is 3.2%. HCV infections progress easily to chronic hepatitis. Twenty to thirty percent of HCV infections lead to liver cirrhosis and hepatocellular carcinoma within two decades. HCV infection has become a main cause of hepatocellular carcinoma and end stage liver disease.HCV is an enveloped virus with a positive-sense RNA genome. The genome contains one centrally located open reading frame (ORF) that encodes structural and nonstructural proteins. Short non-coding regions at each end of the genome (5'NCR and 3'NCR) are required for replication of the genome. Regions of the genome include 5'NCR, C, El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, NS5B and 3'NCR from the 5'terminus to the 3'terminus. The core (C) region encodes a nucleocapsid protein and the E1 and E2 regions encode two envelope proteins. The P7 region and genes from NS2 to NS5 encode nonstructural proteins with various functions.HCV is highly mutable with mutation rates varying among different regions of the viral genome. The 5'NCR and the core region are relatively conserved while the E regions show the highest variation. HCV 5'NCR is the most conserved region in terms of both length and sequence. Rare mutations in this region constitute an important basis for genotyping. Six major genotypes have been identified, each of which further divided into several subtypes. Distribution of HCV genotypes and subtypes varies geographically. The predominant HCV genotype in China's mainland is lb, followed by 2a and low frequencies of la,2b,3b,4a, and 6a. Because sequences in the 5'NCR determine the genotype, it is speculated that variations in this region relates to clinical features, especially the response to antiviral treatment. It is reported that HCV genotypes may account for some of the variance in the clinical course of infection. Patients infected with genotype 1 often demonstrate higher viral loads and lower response rates to antiviral treatment than those with other genotypes. However, the correlation between genotypes and the clinical course is still debated. HCV 5'NCR contains the origin of replication and plays an important role in genome replication and protein translation. Consequently, some mutations in this region might affect the efficiency of viral replication and response to interferon (IFN) treatment.NS5A is a nonstructural protein that functions in RNA replication and maturation of the viral polyprotein. It inhibits apoptosis of the hepatocyte and downregulates the antiviral response stimulated by IFN a. Mutations in certain NS5A sequences may weaken its anti-IFN effect. Researchers from Japan discovered that the sequence from amino acid 2209 to 2248 was related to sensitivity to IFN, and named it the interferon sensitivity-determining region (ISDR). They found that mutations of four or more amino acids in this region resulted in improved response to IFN. However, studies from Europe and America did not confirm the relationship between mutations in ISDR and response to treatment, leading to speculations that there are other sequences in NS5A related to IFN sensitivity. To date, there have been many studies on ISDR but few studies correlating mutations in the entire NS5A region to IFN response. It has not been reported in China.In order to further investigate mutations in the 5'NCR and NS5A regions and their effect on the clinical course, genotypes of HCV in Shandong Province of east China were analyzed using gene chip assays and sequencing. Clinical significance of the mutations was further evaluated in patients receiving interferon therapy. Materials and Methods:1. One hundred and seventy patients with chronic hepatitis and/or cirrhosis due to HCV infection seen in Jinan Infectious Disease Hospital from January 2005 to October 2008 were enrolled. All patients were positive for anti-HCV and HCV RNA. None was co-infected with HAV, HBV, HEV, HIV, or complicated with drug or alcohol-induced liver diseases. Selected hepatitis patients were treated with pegylated interferon a (pegylated IFN a-2a 180ug or a-2b 1.0-1.5ug/kg) combined with ribavirin. Patients with genotype lb were treated with interferon and 1000-1200 mg of ribavirin for 12 months while those with genotype 2a were treated with interferon and 800-1000 mg of ribavirin for 6months. HCV genotypes were analyzed by a DNA chip assay. Relationships between genotype and disease severity, route of infection, viral load, and response to IFN were evaluated.2. One hundred and eighteen chronic hepatitis C patients hospitalized in Jinan Infectious Disease Hospital from January 2008 to December 2009 were enrolled. The 5'NCR of HCV was sequenced. Sequence variations were analyzed with Clustal X 2.0 and Mega 4.0. Phylogenetic trees based on 5'NCR were constructed. Correlations between viral genomic variations and serum levels of HCV RNA as well as response to IFN were investigated.3. The NS5A region of type lb HCV genomes from 35 patients was sequenced prior to combined IFN a and ribavirin treatment. Nucleotide and amino acid sequences of NS5A were analyzed using Clustal X 2.0 and Mega 4.0. Mutations in the entire region and in specific areas (ISDR and V3) were determined by comparing with the HCV lb standard strain (HCV-J). Correlation between variations in NS5A and response to IFN was studied prospectively.Results1. Genotypes 1b,2a, 1a,3b, 1b/2a, and 1a/1b were present in 63.10%,28.57%, 1.19%,1.19%,5.36%and 0.60%of patients, respectively, according to the gene chip assay. No significant difference was observed in the distribution of genotypes between patients with chronic hepatitis and patients with cirrhosis (x~2=2.35, p> 0.05). Neither was there a difference in HCV genotypes between patients infected via blood transfusion and patients without a transfusion history (x2=7.63, p> 0.05). Viral loads were significantly higher in patients infected with genotype 1b than those with 2a (6.42±1.0 vs 5.69±1.15,p=0.0001). SVR (sustained viral response) in genotype 2a infections was higher than in 1b infections.2. HCV genotypes were determined in 118 patients by 5'NCR sequencing. The numbers of cases among genotypes 1b,2a, 1a,3a,3b, and 6a were 65,45,2,1,2, and 3, respectively.3. Sequences of 5'NCR in 42 type 1b HCV genomes were identical to most of genotype lb isolates in Genbank. Twenty three of the sequences showed 1-2 base substitutions. The two characteristic mutations were 120 C-T (9 cases) and 204 C-T (8 cases). Two la strains were identical to the Chinese reference strain and homology to the two American reference strains were 99.5%and 100%. Homology of 5'NCR among type 2a genomes was 97.8%-100%with 11 base substitutions. There were type 2a sequences identical to that of the reference strain. Characteristic mutations were at sites 222 and 247. Type 2a genomes were thus divided into 3 groups according to these sites in 5'NCR, i.e., both T, both C, and C/T. Homology of 5'NCR between the single type 3a strain and the standard strain was 98.1%with 4 base substitutions. Homology of 5'NCR between the two type 3b strains and reference strains was 99.1%and 100%. All three type 6a sequences showed the distinctive CA insertion at site 145 of 5'NCR, with altogether 3 base substitutions when compared with reference strains. Two of the three sequences were identical to one of Chinese strain and homology with another Chinese strain and Hong Kong strain was 99.5%. Homology between the other type 6a strain and the 3 reference strains ranged within 98.5%-99.5%.4. Quantities of serum HCV RNA in patients infected with type 1b variants with a C-T mutation at site 120 of the 5'NCR and in patients infected with the wild strain were 5.16±1.40 log and 6.14±1.01 log, respectively (p=0.041). Quantities of serum HCV RNA in patients infected with lb variants with a C-T mutation at site 204 of 5'NCR were 5.95±0.95 log (p=0.23 when compared with those of patients infected with the wild strain). Quantities of serum HCV RNA in patients with type 2a variants with a C-T mutation at site 220 or site 247 were not statistically different from those of patients infected with the wild strain. The response to IFN did not differ significantly between variants of genotypes 1b and 2a and wild strains.5. Entire sequences of HCV NS5 A were obtained in 20 of 35 patients. Sustained viral response (SVR) was achieved in 11 of the 20 patients, while the other 9 patients experienced no response or relapse (NR). Comparison with the HCV J strain revealed high mutation rates in the NS5A region with substitutions of 136±9.2 nucleotides and 31.3±4.2 amino acids. However, homology of sequences among patients in Shandong province was high. Amino acid substitution numbers within the entire NS5A region were not significantly different between SVR and NR patients (32.4±4.4 and 30.4±3.7, p=0.302). Numbers of cases with mutant-type, intermediate-type, and wild-type ISDR were 1,6,2 among SVR patients and 0,4,7 among NR patients (p> 0.05). Amino acid substitution numbers in the IFN/RBV resistance-determining region (IRRDR) were 6±1.33 in SVR patients and 4.4±1.1 in NR patients (p= 0.014). Therefore amino acid substitution numbers of?6 in IRRDR was predictive of SVR (p=0.04).Conclusions1. The predominant genotype in patients with chronic HCV infection is lb, followed by 2a and low frequencies of la,3a,3b,6a and mixed genotypes. Genotypes 3 a and 6a have not been reported previously in Shandong province.2. Genotype lb is associated with higher viral loads but not disease severity. Blood transfusion is the route of infection in most of the patients. Patients infected with genotype 2a have higher SVR, while the rate of relapse is high in genotype 1b-infected patients.3. HCV 5'NCR is highly conserved. Sequences of 5'NCR display characteristic mutation patterns in both genotypes 1b and 2a. C-T transition at site 120 of genotype 1b impedes viral replication but other mutations are not as significant. Point mutations in 5'NCR do not affect viral response to IFN.4. There are significant sequence differences in the NS5A region between the J strain and HCV strains in Shandong province, even though NS5A sequences in the province are highly homologous. Amino acid substitution numbers of?6 in IRRDR is predictive of SVR. |
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