| Hepatitis B virus (HBV) chronic infection afflicts about93million people in China, who are exposed to the risk of chronic hepatitis B,cirrhosis, liver failure and hepatocellular carcinoma development. In our country, around35million people died of HBV infection-related diseases each year. The anti-HBV therapy is the main treatment of chronic hepatitis B and the block to the development of cirrhosis, liver failure and hepatocellular carcinoma. Nucleos(t)ide analogs (NAs) play an important role in anti-HBV treatment. However, with the extensive and longer use of NAs treatment, the problem of viral resistance has become a thorny issue for clinical anti-HBV therapy. Lamivudine (LAM) listed in1998has more than13years of clinical anti-HBV treatment history in China and more than1.2million patients with chronic hepatitis B had accepted LAM antiviral therapy. Because LAM is easy to take, safety, and the cost of treatment is relatively low, there are still a large number of patients taking the drug.Because of the lack of proofreading activity, HBV polymerase is prone to emerg errors easy in the viral replication process, resulting in a large number of sequence diversity of virus strains and the formation of viral quasispecies. Under the drug selection pressure, drugs adaptable virus variants may evolve as the advantages of strains, causing virus resistance. NAs target the HBV reverse transcriptase (RT) activity and inhibit viral replication. Drug-resistance-associated mutations include primary resistance mutations and compensatory mutations. The former directly decreases the drug susceptibility of the virus and the latter indirectly compensates for the viral replication fitness decreased by the primary drug-resistant mutant virus strains.In terms of LAM resistance, rtM204V and rtM204I are called YVDD and YIDD variation in YMDD motif. rtV173L and rtL180M are well recognized compensatory mutation for rtM204V mutant, and rtL801is a compensatory mutation for rtM204I mutant. LAM has the low genetic barrier to resistance and the high virus resistance incidence. The virus resistance incidence were17%,40%,55%and67%after1-,2-,3-, and4-years LAM mono-therapy, respevtively.Because of the extensive, longer NAs treatment, and unreasonable NAs usage in many cases, emergence of HBV strains with new or potential LAM-resistance-associated mutations increases. In recent years, the new YSDD and the YKDD mutant appeared in patients who received LAM treatment with poor response or no response, which has attracted the attention of schoolars. Therefore, except the detection of classical resistance mutations in clinical antiviral treatment process, identification of potential HBV resistance variation is of great significance for comprehensive understanding of viral resistance and guide the rational adjustment of anti-HBV treatment options.Objective To study virological characteristics of a newly-found mutation M204Q in the reverse transcriptase domain of HBV from chronic hepatitis B patients with LAM treatment failure for a long time, and to identify the association between the rtM204Q mutation and LAM resistance.Methods1. Screening and selecting new or potential LAM-resistant mutations from a large number of sequenced HBV RT genes were completed in Viral Hepatitis Research Laboratory, PLA302Hospital, Beijing. The analysis is performed in combination with clinical information such as NAs usage, virologic and biochemical responses.2. One representative patient’s sample was selected for further study from10cases detected with HBV YQDD mutation. This patient received long-term LAM therapy with failure. Direct PCR sequencing showed that the patient’s sample harbored HBV mutational pattern of rtL180M+rtM204I/Q. The full-length RT region was amplified with nested PCR and the PCR product was cloned into the pGEM-Teasy vector. Forty clones were randomly selected for clonal sequence analysis of drug-resistance-associated mutations.3. Site-directed mutagenesis was performed to convert the pTriEX-1.1-HBV vector and silence its downstream natural Sph I restriction site. After restriction enzyme digestion and ligation procedure, the1.1-ploid genome length HBV recombinant plasmids harboring wild type and four different mutants in RT region were constructed.4. The replication-competent constructs pTriEx-wRT and pTriEx-mRT were transiently transfected into the HepG2cells by Fugene HD transfection reagent, respevtively. Four hours post-transfection, new medium containing different concentration of nucleos(t)ide analogs was applied every other day for4days. To analyze the phenotypic characterization of the HBV mutant under the drug pressure, the supernatant was collected and HBV DNA quantitation was done using real-time PCR. Three independent repeated experiments were performed.Results1. YQDD mutation was detected in10patients’samples in total. The representative patient selected for further study had virologic breakthrough in one-year LAM. HBV DNA load increased from negative to1.88×106IU/mL, Alanine transaminase (ALT) from normal increased to126U/L. HBV RT region was amplified and the PCR product was cloned and sequenced, the genotypic analysis results were the same as the directly sequence. In37clones obtained, thirteen (35.1%) were wild type strain, eleven (29.7%) were rtM204Q strain, two (5.4%) were rtM204I strain, ten (27.0%) were HA181T strain, and one (2.7%) wasrtA181T+rtM204Q.2. Site-directed mutagenesis successfully converted the pTriEX-1.1-HBV vector for the purpose of vector fragment. Xho I/Sph I digestion of HBV RT region of the five mutants and the converted vector, and the two fragments were ligated. The recombinant plasmids were prepared for transfection.3. The1.1-unit recombinant HBV replication plasmids were respectively transfected into HepG2cells. HBV replication force capacity test showed that wild-type strain had replication capacity of (6.00±0.096)×106IU/mL. The replication capacity of rtM204Q, rtM204I, rtA181T, rtA181T+rtM204Q was89.95%,46.28%,55.77%and34.44%compared to that of wild-type strain, respectively.4. The phenotypic characterization of rtM204I/Q was evaluated under four different concentrations of the NAs pressure. Three independent repeat phenotypic resistance experimental results showed that both rtM204I and rtM204Q strains were susceptible to adefovir (ADV), entecavir (ETV), and tenofovir (TDF). By contrast, both strains exhibited resistance to LAM to different extents. The rtM204I strain and the rtM204Q strain had a1395-fold and75.7-fold decrease of susceptibility to LAM compared to the wild-type strain by measurement of50%inhibitary concentration (IC5o), respectively.Conclusions 1. YQDD mutation was detected in the YMDD motif that has been proved to be highly associated with LAM resistance from10LAM-treated patients (part of them were LAM-refrectory), suggesting that this mutation is associated with LAM therapy and failure of treatment.2. The rtM204Q mutant HBV strain had a lower viral replication capacity compared to wild-type strain, but had a higher viral replication capacity compared to classic LAM-resistant rtM204I strain.3. The rtM204Q mutant HBV strain had resistance to LAM, but resistant extent is not lower compared to M204I strain. Both mutant strains remain sensitive to ADV, ETV, and TDF.4. Taken together, rtM204Q is a novel LAM-resistance-associated HBV mutation that has not been reported previously. This mutant stain could coincide with classic LAM-resistant strains in LAM-refectory patients. The study furthers our knowledge of HBV drug resistance and offers valuable clue to help clinicians manage LAM-resistant HBV infection. |
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