Establishment of a method to detect duck hepatitis B virus covalently closed circular DNA based on rolling circle amplification / 病毒学报

Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.

Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV / 病毒学报

Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.

Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA).</p><p><b>METHODS</b>HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA.</p><p><b>RESULTS</b>The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases.</p><p><b>CONCLUSION</b>The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.</p>

Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis.</p><p><b>METHODS</b>HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples.</p><p><b>RESULTS</b>All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells.</p><p><b>CONCLUSION</b>The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.</p>