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Abstract
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Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (epsilon) located in the preCore region, essential for viral replication. epsilon stability is enhanced by the presence of preCore variants and epsilon is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the epsilon signal was found to be the main constraint for selecting main preCore mutations. Analysis of epsilon-signal variability suggested the essential nature of the epsilon structural motif and that certain nucleotides may be involved in epsilon signal functions. |
