| Currently, there are more than 350 million people with chronic hepatitis B virus(HBV) infection, including 93 million people in China. Chronic HBV infection could cause chronic hepatitis B (CHB), live cirrhosis, liver failure and liver cancer. Two major kinds of anti-HBV agents have been used for the treatment of HBV infection, i.e.,α-interferon (IFN-α) and nucleoside/nucleotide analogues. The role of IFN-αis mainly via inducing host cells to produce antiviral proteins and regulating immunity against HBV. The virologic response of IFN-αis longer than that of nucleos(t)ide analogues, but clinic response of IFN-αis not high which is around 30% to 50%. Furthermore, there are many side effects and high costing. The other important anti-HBV agent is nucleos(t)ide analogues.Nucleos(t)ide analogues can be catalysised by the phosphokinase to form 3-phosphorylation compounds, which can compete with natural deoxyguanosine triphosphate(dGTP)to bind with HBV. The target of nucleos(t)ide analogues is HBV polymerase/reverse transcriptase (RT). Nucleos(t)ide analogues could inhibit HBV replication in three steps: base priming, negative strand synthesis by hnRNA reverse transcription, and positive strand synthesis. In our country, four nucleos(t)ide analogues are used in clinic. i.e., lamivudine (LAM), adefovir (ADV), entecavir (ETV), and telbivudine (L-dT). HBV has high frequency genetic variation because its reverse transcriptase is lack of proof reading function. The variation rate of HBV is about 10-7 bp/d,significantly higher than the average error rate of DNA viruses. During long-term clinical use of the nucleos(t)ide analogues, HBV stains with drug-resistant variation could be selected from HBV quasispecis pool to become dominant strains and cause disease relapse.HBV resistance sequentially emerges as follows. 1. genotypic resistance, presenting as the emergence of viral populations bearing amino acid substitutions in RT region of the HBV genome that have been shown to confer resistance to antiviral drugs in a phenotypic assay during antiviral therapy. 2. virologic breakthrough, presenting as serum virus load rebound >1 log10. 3. biochemical breakthrough, presenting as the elevation of alanine aminotransferase and/or increase of liver histologic impairment. Phenotypic analysis is an essential tool to identify whether a variation in HBV RT region is associated with drug resistance. It bases on the comparison of HBV replication capacities of variants and wild-type counterparts by cell-transfer of individual viral genomes followed by quantitation of HBV replication intermediates in the presence of antivirals in different concentrations. However, due to complex methodology and the classical Southern blotting assay has low reproducibility, only few of laboratories in China primarily set up the for phenotyping HBV drug resistance.Objective To establish a practical assay of phenotypic analysis for HBV drug resistance to help identify novel and complex drug-resistance-associated variation from patients. In concrete:1. To construct the recombinant vectors containing 1.1-fold genomes of HBV wild-type and LAM-resistant mutant strains.2. To determine resistance phenotype of mutant virus by measuring viral replication levels of wild-type and mutant viral genomes after transfecting them into HepG2 cells and culturing the cells in different concentration of the drug.3. To develop real-time polymerase chain reaction (PCR) assay for determination of HBV replicative intermediate level and compare with the classical Souther blotting assay.Method 1. HBV DNA was extracted from serum of a CHB patient resistant to LAM. The resistance mutational pattern was rtL180M+rtM204V. HBV DNA RT gene was obtained by nested PCR. The mutant viral gene was numbered as 9077 and cloned into pGEM-Teasy vector. The plasmid pGEM-Teasy-RT vector was termed as 9077-p-m. Corresponding wild-type viral gene was produced by site-specific mutagenesis and the recombinant pGEM-Teasy vecter was termed as 9077-p-w.Plasmid vectors 9077-p-m, 9077-p-w and pTriEx-HBV(C) were digested by Xho I/Nco I to construct the recombinant vectors of wild-type HBV pTriEx-wRT(S9077-1) and LAM-resistant mutant strain:pTriEx-mRT(S9077-2). pTriEx-wRT(S9077-1) and pTriEx-mRT(S9077-2) were transfected into HepG2 cells. HBsAg and HBeAg levels in the supernatant were measured by ELISA.2. pTriEx-wRT(S9077-1) and pTriEx-mRT(S9077-2) were transfected into HepG2 Cells. Various concentrations of LAM (0μmol/L, 0.01μmol/L, 0.1μmol/L, 1μmol/L, 10μmol/L, and 100μmol/L) were added 60 hours after transfection. Intracellular HBV DNA was extracted 5 days after continuous LAM treatment. Quantitative real-time PCR and Southern blotting analysis were performed to detect the HBV DNA level.Result 1. HBV RT gene cloned from the LAM-resistant patients was confirmed containing correct mutational pattern by cloning sequencing. After site-specific mutagenesis, the HBV RT gene successfully became the wild- type. After transfection of recombinant plasmids containing either LAM-resistant or wild-type HBV 1.1-fold genomes into HepG2, the expression levels of HBsAg and HBeAg in the supernatant reached anticipated levels.2. After adding various concentrations of LAM, intracellular HBV replicative intermediate level decreased significantly along with the increase of drug concentration. The IC50 was 0.04±0.01μmol/L for the wild-type strain. By contrast, no such obvious decrease of HBV replicative intermediate level was observed in the cells transfected with HBV genome containing LAM-resistant rtL180M+M204V mutations. The IC50 was >100μmol/L, which was about 2500-fold higher than that of the wild-type HBV strain.Conclusion 1. The study constructed recombinant vectors containing 1.1-fold HBV wild-type or LAM-resistant HBV RT genes. After transfecting the recombinant vectors into HepG2, the recombinant vectors could perform high levels of HBsAg and HBeAg in the supernatant.2. The study take the transfection with liposomes in vitro to research HBV DNA drug resistance. Successfully established the method for accessing drug susceptibility of HBV in vitro. This phenotyping assay will help to identify drug-resistance-associated variants detected from patients. |
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