Research On The Detection Of HBV Gene Mutation Induced By Nucleoside/Nucleotide Analogues And Clinical Significance In Chronic Hepatitis B Patients

Chronic hepatitis B is a significant public healthy problem wordwide. At present, it is estimated that more than 350 milloin individuals are chronically infected with HBV. HBV infection can cause acute, fulminant or chronic disease, liver cirrhosis, even progress to hepatocellular carcinoma. According to the figures of World Health Organization, 1.2 million hepatitis B patients die of diseases related to HBV infection every year.HBV is a number of the Hepadnaviridae. HBV DNA has a non-completed circular double stranded DNA of 3200bp and four patially overlapping open-reading frames: pre-S/S, pre-C/C, P and X regions.S region encodes pre-S1, pre-S2 protein and HBsAg; C region encodes HBeAg and HBcAg; P encodes DNA polymerase (DNAP), and X region encodes X protein. Because HBV replicates rapidly, and DNA poly- merase lacks of revise mechanism, HBV is characteristic of heterogeneity and quasispecies.Nowadays, there are six drugs which were approved to treat hepatitis B: IFN-α, PegIFN-α2a, nucleoside/nucleotide analogues such as lamivudine, adefovir dipivoxil, entecavir, telbivudine. The significant adverse reactions of IFN limit its application. Nucleoside/nucleotide analogues are potent viral suppressors and have high response rate, can decrease ALT and HBV DNA levels and improve liver histology significantly, but none is able to permanently eradicate HBV,so it is necessary to use for long-terms. Prolonged use of nucleoside/nucleotide can cause the emergence of drug-resistant mutants and lead to reccurance of hepatitis, even fatal liver failure.Thus, it is necessary to detect occurrence of drug-resistance closely in the course of treatment with nucleoside/nucleotide analogues, in order to modify therapeutic regimen in time. At the same time, due to various nucleoside analogues can be cross-resistant and HBV may have primary resistance and the phenomenon of quasispecies, but at present, the approach of detecting virus drug-resistance can detect only one site, hence we need badly one approach which can detect multi-sites mutations at the same time, to guide clinical treatment. to guide the physician choose reasonable medication, and reduce the formation of drug-resistant strains, lower t the treatment costs of patients.Our research desire to develop one approach which can detect multi-sites mutations at the same time and find new mutation sites, through analysing mutations of amino acides and bases in HBV DNA part fragment of polymerase region by PCR amplification, molecule cloning technique and sequencing, meanwhile, we studide the amino acid variation in HBV P region in patients with application of Lamivudine, adefovir dipivoxil, and the two drugs co-treament, and the quasispecies of HBV in some patients, to verify the common variant sites of nucleoside drugs and explore new variation site, confirmed the existence of HBV quasispecies.Results: (1) Our research obtained amino acid sequences between 152 and 294 site in P region, so it can detect 143 amino acides changes at the same time. The results can provide evidence for clinical doctors choosing the optimal therapeutic regimen. (2) lamivudine causes rt180,rt204 sites mutations, while rt236,rtN238H/K variation occur in the course of adefovir dipivoxil treatment. Different patient has individual amino acid substitution. (3) After rt180 and/or rt204mutation, the effection of treatment is not obvious if we continue to use lamividine;but adding adefovir dipivoxil, the condition can be improved obviously; The effection of lamividine combines with adefovir dipivoxil is poorer in rt181 mutation than not; After rt236 mutation, keeping on adefovir dipivoxil treatment can't improve patient's condition prominently. (4) The homology of HBV DNA among different clones from the same patients exceeds 95%, some clones may occur base deletion mutation.Conclusion: (1) our research found one approach can detect multi-sites mutations simutanously. (2) we validated the common mutation sites in the course of lamivudine and adefovir dipivoxil treatment, rtN238H/K may related with gene mutation caused by adefovir dipivoxil. (3) We observed the therapeutic efficacy of continuing and changing remedia after HBV gene mutation induced by nucleotide (nucleoside) analogues. (4) Different patients has individual amino acid substitution. The same patient can be infected by different HBV virus strain. (5) Polymerase region of HBV DNA may emerge base deletion in some patients.