Influence Of Specific Variations In HBV Reverse Transcriptase On Nuleotide Analogs Susceptibilities

Hepatitis B virus (HBV) infection represents a global public health problem. Nucleotide analogs have been broadly used in therapy for chronic hepatitis B patients. A problem occurred during long-term management with nucleot(s)ide analogs is the emergence of drug resistance. An established cause of drug resistance is the occurance of specific mutations in the reverse transcriptase of HBV. For the research related to drug resistant mutations, susceptibility assay of HBV isolates in vitro plays an important role. For the lack of a robust infectable cell system, susceptibility assay currently rely on the transfection of recombinant HBV DNA into hepatoma cell lines. Therefore a replication-competent HBV DNA recombinant becomes critical under these circumstances. Clone of clinical HBV isolates using traditional digestion-and-ligation technique in a replication-competent way can be very difficult because of the sequence heterogeneity and the unique structure of HBV genome. In the present study, we developed a new strategy for constructing HBV 1.1×recombinant. The core of this strategy is a fragment replacement method named Fragment Substitution Reaction (FSR). FSR allowed us to clone PCR fragments without digestion-and-ligation, providing a new tool for cloning fragment or genome amplified from serum HBV DNA, making the research on HBV phenotypic assay more convenient. Using this strategy, we performed phenotypic assays for HBV strains carrying an RT S246T variation or an RT N118H isolated from chronic hepatitis B patients with partial response to ETV and ADV therapy respectively. Susceptibility data indicated that these strains are still sensitive to ETV and ADV in vitro respectively.Objective To develope a new strategy for constructing 1.1-genome -length HBV DNA recombinants supporting HBV replication in vitro and to perform phenotypic assay for HBV isolates cloned from patient sera.Methods1. A new strategy for constructing HBV1.1×genomes recombinants based on the technique named Fragment Substitution Reaction was developed.2. HBV DNA was extracted from the serum of a CHB patient who did not respond very well to ETV therapy. ETV susceptibility assay was performed using an HBV1.1×recombinant containing the HBV DNA derived from the patient by transfecting it into HepG2 cells.3. HBV DNA was extracted from the sera of CHB patients who did not respond very well to ADV therapy. HBV1.1×recombinants containing the HBV DNA derived from the patients were constructed. One of the HBV1.1×recombinants was stably transfected into HepG2 cells and ADV susceptibility assay was performed using the stable cell line.Results1. Fragment Substitution Reaction allowed us to clone a DNA fragment into a vector effectively without digestion and ligation;2. HBV1.1×recombinant constructed through Fragment Substitution Reaction method can replicate HBV in vitro;3. The HBV isolate carrying RT S246T variation yields an ETV IC50 of 0.99±0.09 nM;4. The HBV isolate carrying RT N118H variation yields an ADV IC50 of 0.076±0.010μM. Conclusion A new strategy for constructing replication-competent HBV1.1×recombinant from clinical HBV DNA was developed. The core of this strategy, a fragment replacement method named Fragment Substitution Reaction, provides a robust tool for cloning fragment or genome amplified from clinical HBV DNA, making the research on HBV phenotypic assay more convenient. Using this strategy, we performed phenotypic assays for HBV strains carrying an RT S246T variation or an RT N118H isolated from chronic hepatitis B patients with partial response to ETV and ADV therapy respectively. Susceptibility data showed that these strains are still sensitive to ETV and ADV in vitro respectively, suggesting that the partial response observed in the patients studied cannot be attributed to the varitations at RT aa246 and aa118 of HBV.