Characterization Of High Replicative Adefovir-resistant Hepatitis B Virus Isolates With Presl Deletion From A Patient Receiving Sequentiallamivudine And Adefovir Therapy

More than350million of the world population are infected with Hepatitis B virus (HBV), thus, HBV infection is still a serious health problem worldwide. Chronic hepatitis B virus infection accounts annually for million deaths worldwide from cirrhosis, liver failure, and hepatocellular carcinoma (HCC). The treatment of chronic hepatitis B remains a clinical challenge. Nucleoside and nucleotide analogs (NAs) including Lamivudine (LAM), Adefovir (ADV), Entecavir (ETV) and Tenofovir (TDF) act on the reverse transcriptase (RT) of HBV and are effective inhibitors of HBV replication, can suppress viral replication and delay the process of liver disease. However, they generally must be used for long periods to maintain effectiveness. The prolonged use of antiviral drugs is associated with the emergence of resistant HBV strains which are responsible for therapeutic failure and progression of liver disease. HBV mutants frequently arise due to the error-prone character of reverse transcription. Under the selection pressure, drug-resistant HBV mutants are more likely to become the dominant strain. Therefore,when virological breakthrough occurred in the clinical treatment process,we should do further analysis of its drug resistance. Phenotypic analysis for drug susceptibility is an important tool for researches on the resistant HBV isolates and for the evaluation of the effect of new antivirals. According to the study of clinical HBV mutants, a reliable system that can reflect the biological function of mutants in vivo should be developed. This work can be divided into two parts below. Part OneObjective:To construct an appropriate expression plasmids containing the whole HBV information of clinical isolates and sequence analysis.Method:Select serum samples from chronic hepatitis B (CHB) patients who experienced a viral breakthrough in the process of treatment of lamivudine and adefovir sequentially. The RT region was amplified by PCR using HBV DNA extracted from sera as the template. PCR products were then sequenced and analyzed. Full-length HBV genome was amplified from a special serum sample and cloned into PUC19. Direct sequencing was carried out on each clones. The dominant strain was cloned into vector PHY106to construct a recombinant plasmid containing the1.1unit of HBV genome. The same, wild-type full-length HBV genomes were used to construct the recombinant expression plasmids PHY536207(genotype B) and PHY97(genotype C) respectively.Result:Six clones containing Full-length of HBV genome were constructed. Sequencing analysis found the strain isolated from the patient was very special with a207-bp deletion in the preSl region and with rtN236T and/or rtA181T substitution. The expression plasmids containing the1.1unit of HBV genome were constructed successfully for the vitro cell experiment.Conclusion:The recombinant constructs could be constructed successfully with clinical isolates from serum.Part Two:Objective:Characterization analysis of the clinical mutant isolates conducted with recombinant expression plasmid in vitro transfection.Method:The recombinant expression plasmid containing the1.1unit of HBV genome constructed in part one was transfected into Huh7cells. The plasmids from genotype B wild-type, genotype C wild-type and clinical isolates were compared in one transfection. The supernatant was selected and HBsAg and HBeAg expression were determined by ELISA. Intracellular replicative HBV DNA level was determined by Southern blot. Phenotype of drug sensitivity including lamivudine, adefovir, entecavir and tenofovir was identified by Southern blot.Result:Southern blot analysis confirmed that the recombinant expression plasmid from clinical isolates produced more intracellular replication intermediate DNA than wild-type HBV in transfected Huh7cells. Phenotypic analysis then showed significantly decreased susceptibility to adefovir, a mild decrease in susceptibility to lamivudine and Tenofovir, remained sensitive to entecavir.Conclusion:Despite the HBV gene deletion and resistant mutation, higher replication competence was detected. Thus, mutation under the drug pressure probably enhance replication ability of HBV. It may be related to selection pressure of antivials. Drug susceptibility analysis in vitro is consistent with clinical manifestations.