| Background: Antiviral therapy is the most important for treating patients with chronic HBV infection-associated diseases. With the availability of increasing kinds of nucleos(t)ide analogs (NA), clinicians have more options in anti-HBV strategies. However, long-term use of NA may lead to the development of drug resistant strains. The selection of drug-resistant mutants is a dynamic process and the duration from detectable genotypic resistant viruses to clinical phenotypic resistance (i.e., virologic breakthrough and then biochemical breakthrough) largely depends on replication capacity of the mutants. Therefore, detection of resistant mutants and analysis of their replication capacity may help clinicians to monitor drug-resistance and tailor anti-HBV strategies adapted to the viral features of individual patients.Objective:To monitor the genotypic resistance in order to help clinicians to optimize the antiviral treatment. To evaluate the influence of replication capacity of HBV mutants that developed during antiviral therapy, especially for those harboring non-classic RT mutations that potentially associated with development of resistance. To facilitate the methodology of phenotypic assay in order to extend its application in clinic.Methods:Firstly, genotypic assay was performed in 6000 patients with chronic HBV infection. Five patients with chronic hepatitis B were followed up for at least 50 weeks, and their serum HBV DNA plus liver function such as alanine aminotransferase (ALT) were monitored dynamically. Secondly, the full-length HBV genomes of 6 patients were cloned into pGEM-Teasy vector, altogether 11 clinical isolates harboring various mutation patterns were obtained, and other 19 mutants were generated with site-directed mutagenesis in order to make convenient comparisons between the mutants and wild-type strains. Thirdly, a SYBR Green I real-time PCR was developed to evaluate the replication capacity of various mutants, which had been proved to be a practicable assay with high sensitivity and specificity. Lastly, the viral replication was investigated using a vector-free transfection assay. Replication capacity of these mutants was evaluated by our real-time PCR. To verify the results, the Southern blot analysis was applied to quantitate the HBV replicative intermediates derived from patient #624.Results:(1) Based on the results of our genotypic assay, the antiviral treatments of most enrolled patients were optimized by their clinicians.(2) The qPCR assay had a high sensitivity and controllable variability for quantitating HBV replicative intermediates DNA, its lower detection limit reached 200 IU/mL, and its dynamic range was 6 Log10 units of magnitude.(3) Compared to wild-type counterpart, mutant rtL217P produced 1.98-fold higher replicative intermediate level, and mutant rtM204I+rtL217P increased the replication level to 1.20 fold. Other mutational patterns (rtV173M, rtA181S/V, rtM204I, rtQ215H, rtL229M, rtN238H, rtV84M+rtA181S+rtM204I, rtV84M+ rtM204I, rtA181S+rtM204I, rtA181V+rtL229M, rtQ215H+rtN238H) reduced viral replication capacity to different extents, respectively. The results of mutants derived from #624 patients were consistent with Southern blot analysis.(4) Among 6000 patients,6.57% (394/6000) showed mutations in the rt229 locus. The frequency of rt229 mutations was higher in LAM-treated than other patients (including ADV-treated, naive, and antiviral-treatment-unclear patients) (78.93% vs.21.07%). The most common mutation pattern was rtL229V±LAM. Viral replication assay showed that the rt229 substitution reduces wild-type HBV replication, but enhances the replication capacity of LAM-resistant mutant.Conclusions:(1) We recommend that patients with chronic HBV infection should be monitored by genotypic assay routinely to intervene antiviral treatment early.(2) The study provides novel information for replication features of mutant strains, especially, the rtL217P and rt229 substitution might be the novel compensatory mutations associated with LAM resistance.(3) The study offers a practical assay for evaluating HBV replication and valuable information of replication features of mutant viruses, which may help analyze drug-resistant phenotype and estimate duration from genotypic resistance to phenotypic resistance in clinic.
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