| Chronic hepatitis B(CHB) is a potentially life-threatening liver disease caused by hepatitis B virus(HBV). It is a significant global health problem. It can cause chronic liver disease and chronic infection and puts people at high risk of death from cirrhosis of the liver and liver cancer. Approved treatments in China for CHB include interferon(IFN) and nucleos(t)ide analogues(NAs)(lamivudine, adefovir dipivoxil, entecavir and telbivudine). Although NAs treatment are more convenient than IFN-based therapy and have fewer side effects and faster HBV DNA decline. However, a long duration of NAs treatment is associated with an increasing risk of development of drug resistance. Antiviral resistance and poor adherence are the most important factors in treatment failure.The discrepancy of the potential antiviral resistance mutation profiles within HBV reverse transcriptase(RT) between NAs-untreated and-treated patients with CHB have not been well characterized. Thus, in the first chapter of this research, full-length HBV RT sequences from 169 NAs-untreated and 131 NAs-treated CHB patients were amplified by sn PCR, sequenced by PCR product-based direct sequencing or clone sequencing, and analyzed by bioinformatics method. Patients were found with either B-genotype(76.7%, 230/300) or C-genotype(23.3%, 70/300) infections. Between NAs-treated and-untreated group, no significant differences were observed in B- to Cgenotype ratio and gender. However, a significantly higher age, HBV DNA level and HBe Ag positive rate were found in NAs-untreated group. Genotype C displayed higher mutation frequency and number of viral genomes with mutation than genotype B. Single or multiple mutations in the RT region of HBV polymerase were detected in 155 CHB patients among 300 CHB patients. Overall, serum HBV DNA level were significantly lower in patients with multiple mutations compared to patients without mutations and those with single mutation. Similar results in terms of viral repation were obtained statistical significance were reached in HBV B or C patients. Moreover frequency of HBe Ag positivity was higher in the absence of mutations compared with the presence of a single substitution or of multiple mutations. Patients with multiple mutations were mainly infected with genotype C.Among the screened 42 potential NA resistance(NAr) mutations, only 17 polymorphic sites were identified and 13 novel mutations were detected in 11 positions. Of note, among these novel sites, the prevalence of rt I187 V and rt N226 H were significantly high in NAs-treated group. The known primary drug resistance and secondary/compensatory mutations were only identified in NAs-treated patients at 7 sites. The putative antiviral resistance and pretreatment mutation were found at 10 sites including 11 amino acid substitutions in these two groups. Among the primary drug resistance and secondary/compensatory mutations, there were 4 mutant sites associated with LAM treatment(204, 80, 173, 180) and another two sites(181, 236) associated with ADV treatment. For putative antiviral resistance and pretreatment mutation, there were four mutant sites associated with ADV as their mutation rate in ADV monotherapy was significantly higher than the other three treatment regimes, there were 191, 213, 214 and 221. Another mutant site 229 were associated with LAM as there were 13 among 14 rt L229 were found in LAM-containing therapy and along with the LAM resistance mutation rt M204V/I. These results above suggested that NAs therapy not only offer a major selection factor for the primary drug resistance and secondary/compensatory mutations but also prompt the change of some of the putative antiviral resistance mutations.In this study, we also found that some HBV NAr related mutations have preference in HBV genotype. For example, rt L180 M were mainly found in genotype C while rt L80 V were only found in genotype B. Moreover, rt M204 I and rt M204 V preferred using rt L80 V and rt L180 M as complementary mutation respectively.Furthermore, the frequency of patients carrying multiple mutations was significantly higher in LAM and ADV sequential therapy when compared to the other three treatment regimes. In LAM and ADV combined therapy, only LAM resistant-related mutations were found. However, in LAM and ADV sequential therapy, we not only detected LAM resistant-related mutations but also found ADV resistant mutant rt A181 T. This suggested that LAM and ADV sequential therapy may increase the probability of selection of ADV resistant mutant.In conclusion, these results might provide insights into understanding the evolution and selection basis of NAr mutations under antiviral therapy and choose better treatment regime. The antiviral resistance potency of putative antiviral resistance and pre-treatment mutation call further in vitro phenotypic assay to identify.The second chapter of this research mainly focused on the new mutations found in the first chapter. We aimed at constructing in vitro phenotypic analysis system to evaluate the effect of a high prevalence mutation on antiviral drug resistance and assessing the replicative capacity and susceptibility of rt I187 V alone or in conjunction with lamivudine(LAM)-resistant mutations(rt M204 I, rt L180M/rt M204V) to LAM and ADV in vitro. Replication-competent HBV constructs containing rt I187 V and combined with LAM-resistant mutations were generated using site-directed mutagenesis and using WT and LAM- or ADV-resistant clones as negative and positive control respectively, and followed by trasciently transfected to Hep G2 cells to carry on drug susceptibility assay. At last, intracellular HBV DNA and extracellular virions were detected. In a cohort of 300 CHB patients, 8.7%(26/300) showed substitutions in the rt I187 with V. As a result, rt I187 V were found not only in NAs-untreated group but also in NAs-treated group. Moreover, 15 rt I187 alone were found in NAs-untreated group and 10 rt I187 combined LAM resistant mutation were identified in LAM-containing group. Of note, the prevalence of rt I187 V in genotype B was significant higher than in genotype C. In vitro phenotypic assays showed that the viruses bearing the rt I187 V had impaired replication efficacy when compared to WT and the virus carrying the rt I187 V combined with LAM-resistant single or double mutations showed even more significantly impaired replicative capacities. Furthermore, rt I187 V remained susceptible towards treatment with LAM or ADV in vitro whereas the combination of rt I187 V substitution with LAM-resistant mutations was resistant to LAM but still sensitive to ADV.In conclusion, our study reveals that rt I187 V substitution in the HBV polymerase frequently occur in CHB patients particular with genotype B. However, the emergence of rt I187 V substitution significantly impair viral replication but without affecting drug sensitivity in vitro. Thus, rt I187 V substitution may represent background polymorphisms rather than resistance mutation. Therefore, for CHB patients with rt I187 V combined LAM resistant mutation, we recommend ADV treatment in order to reduce the probability of selection of compensatoty mutation of LAM on the basis of no compensatory mutation emerging. |
