| Four hundred million people worldwide are chronically infected with hepatitis B virus (HBV). Chronic infection consists of three well defined phases: an initial immune tolerance phase, followed by an immune clearance phase and a third non-replicative phase. The HBV genome comprises a partially double-stranded 3.2kb DNA with four open-reading frames (ORF). In the course of virus genome expression, four transcripts are produced. Expression of the four transcripts is directed by the enhancer II/basic core, large surface antigen (L), major surface antigen (S), and enhancerI/X gene promoters. In the replication course of HBV, viral reverse transcriptase (rt) lack a proofreading function and are thus inherently error prone. As a result, HBV strains exist in the host as heterogeneous mixtures known as quasi-species. HBV may not be directly cytopathic. It is believed that the immune function plays a key role in the severity of HBV disease, The immune response to HBV-encoded antigens is responsible both for viral clearance and for disease pathogenesis during this infection. The nucleotide substitutions in HBV genome will change the target epitopes of some HBV proteins, and may have effect on the development of disease. The analysis on full sequences of HBV genome was reported in different countries and in different genotypes, in the course of disease from asymptomatic (ASC) to chronic hepatitis B, in patients with hepatocellular carcinoma (HCC) and in the exacerbation stage of HBV infection. We studied the full sequence of 62 cases HBV genotype C, which is more popular in Asian countries and with poor prognosis in all eight genotypes, and we demonstrated the effect of viral factors on the development of diseases. Accordingly, 62 cases were divided into three groups, 12 ASC were classified as group A (ASC1-12), 37 HBeAg-positive chronic liver diseases (CLD) as group B (CLD1-37), and 13 HBeAb-positive CLD as group C (CLD38-50). Meanwhile, we analyzed the full sequence of patients with the development of HCC, and tried to find out if there had viral factors contribute to the development of HCC besides the host factors. We also analyzed the full sequence of patient receiving the therapy of lamivudine, with the results of persistent respond (group A), with the breakthrough (group B) and non-respond (group C), compared the sequence before and during the therapy in order to find the predicator factor of the viral genome to the therapy. The results are as following: 1. Phylogenetic tree based on the 62 HBV isolates showed that there are two major groups of genotype C1 (3/62) and genotype C2 (59/62). 2. We analyzed the nucleotide substitutions against genotype C2. The nucleotide substitutions in the full sequence were compared among three groups. Between group B (35.2±12.4nt) and group A (27.2±7.7 nt), significant difference was observed (P<0.05), however between group C (42.3±9.4 nt) and group A, more obvious difference was observed (P<0.001), between group C and group B, significant difference was not observed (P>0.05). 3. In the preC/core region, the nucleotide and amino acid substitutions in group B and group C,comparing with group A, there was significant difference (P<0.05 and P<0.001, respectively), this kind of difference was obvious between group C and group A, and comparing group B with group C, there was also had significant difference (P=0.001). In the preC/core region, the mutations in G1896A were detected in 12 cases of 37 cases group B (32.4%) and 8 cases of 13 cases group C (61.5%), none of group A, but the positive detection rate of G1896A did not have significant difference between group B and group C (P>0.05). The mutations in G1896A also existed in 32.4% HBeAg-positive CLD, these patients have lower titer of serum HBeAg (41.1±67.7COI) being compared with that of having wild type 1896 HBeAg-positive CLD (408.7±613.1COI) (P<0.05). We also detected some non-synonymous mutations in the N-terminal of core protein, some of these were within the HLA I-class CTL epitope (codon 18 to 27).4. In the preC/core region, G1896A were detected in 10 cases of 18 cases with persistent response, in 4 cases of 7 cases bearing breakthrough, in 2 cases of 9 cases without response respectively in 34 cases receiving the therapy of lamivudine, no significant difference is showed in three groups (P>0.05). 5. In the X region, the nucleotide and amino acid substitutions between group A and group B, group C significant differences were observed (p<0.05, p<0.001, respectively), but between group B and group C, significant differences were not seen (P>0.05). The X region overlapped with the basic core promoter (BCP), the hot mutations of A1762T, G1764A were detected in 28 and 30 of group B, in all of group C, in 3 of group A, respectively. The mutation in C1653T were detected in 6 of group B and group C, respectively. The mutation in T1753C/G/A were detected in 6 cases of group B and 5 cases of group C, respectively. The mutation of C1653T and T1753C/G/A were detected in different samples, but the samples with these two mutations were accompanied by the mutations of A1762T, G1764A, the mutation of C1653T and T1753C/G/A were not detected in group A. The nucleotide substitutions in above BCP were accompanied by the amino acid change in the X protein region. The mutations in C1653T, T1753C/G/A, A1762T, G1764A were accompanied by the change of codon 94 (from histidine to tyrosine), 127 (from isoleucine to threonine or alanine), 130 (from lysine to methionine), 131 (from valine to isoleucine), respectively. 6. In the X region, the nucleotide substitutions in T1753C/G/A were detected in 6 cases of chronic hepatitis and HCC stage, respectively in 13 cases with the development of HCC. This substitutions were detected in 11 cases of 62 cases, 5 cases in these cases with the development of HCC, this substitution had significant difference in cases with the development and without the development of HCC (P<0.05). A1762T and G1764A were detected in 11 cases and 12 cases of chronic hepatitis stage, respectively in 13 cases with the development of HCC. 7. In the X region, the changes of codon 5 were detected in 12 cases of group A (7 cases to methionine, 5 cases to leucine), 2 cases of group B (1 cases to methionine, 1 cases to leucine), 3 cases of group C (all to leucine),respectively in receiving the therapy of lamivudine, but there showed no significant difference among three groups (P>0.05). 8. In the regulatory element region, in the enhancer I region, the nucleotide substitutions did not have obvious difference among the three groups of 62 cases (P>0.05). Similarly, in the enhancer II, X promoter, large surface promoter and major surface antigen promoter, the nucleotide substitutions in three groups, obvious difference were not detected. But in the core promoter, the nucleotide substitutions in group B (3.2±1.3nt) and group C (3.9±0.9nt) compared with group A (1.7±1.0 nt), significant difference were detected (both P<0.001), between group B and group C, there did not have significant difference (P>0.05). 9. In the X promoter, T1341C/A were detected in 5 cases chronic hepatitis and 5 cases of HCC in 13 cases with the development of HCC, respectively, this nucleotide substitutions were detected in 7 cases of 62 cases, there were 4 cases with the development of HCC in these cases, there had significant difference between with and withous the development of HCC (P<0.05). 10. The deletion were detected in the core promoter, core region, the preS1 and preS2 region. In the core promoter, the deletion region was between 1755 and 1774 nt. In the core region,the deletion were from 2137 to 2306 nt. This will cause the deletion of core region from codon 79 to 136, this region includes the major target of CTL, and could be relative to the immune escape.The deletions in the preS2 were mainly clusted in the amino-terminal of preS2 protein. In 13 cases with the development of HCC, The deletions in the preS2 were detected in 6 cases chronic hepatitis and 3 cases of HCC, respectively, were also mainly clusted in the amino-terminal of preS2 protein. 11. In all samples, the insertion mutation from 1820 to 1838 (CTTTTTGAGCTCCGGAGCT) was detected in only 1 sample (ASC12). 12. The deletion mutations in BCP were also detected in two samples of group A in 62 cases, who developed into CLD after five years. As to these two cases of ASC, we also detected the sequences with CLD phase, and compared with the sequence in ASC, to ASC (7), in CLD phase, and found that this deletion also existed, but to ASC (8), this deletion disappeared and the mutation in 1762 and...
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