A conceptual framework for dynamics of cccDNA in hepatitis B virus / 中华肝脏病杂志

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.

Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.</p><p><b>METHODS</b>Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.</p><p><b>RESULTS</b>Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.</p><p><b>CONCLUSIONS</b>A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.</p>

The biological function of auto-induced expression of the hepatitis C virus soluble core protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.</p><p><b>METHODS</b>The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.</p><p><b>RESULTS</b>The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.</p><p><b>CONCLUSION</b>Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.</p>

In vivo pharmacokinetics of major bioactive components from Tanreqing Injection in rats / 中草药

Objective: To investigate the in vivo pharmacokinetic characteristics of Tanreqing Injection in rats. Methods: Using baicalin (BC), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA) in Tanreqing Injection as biomarkers to characterize the in vivo pharmacokinetics. Rats were received an iv administration of Tanreqing Injection in single dose (1.25, 2.5, and 5.0 mL/kg) or multiple doses (5.0 mL/kg), and the blood concentration of BC was determined using LC-UV method, and the concentration of UDCA and CDCA was determined using LC-MS method. The pharmacokinetic parameters were analyzed by WinNonlin 6.3 software. Results: After a single iv administration of Tanreqing Injection (2.5 mL/kg) to rats, the values of t1/2 for BC, UDCA, and CDCA were (48.19 ± 12.74), (56.19 ± 33.33), and (109.96 ± 58.39) min, and AUC0-6h were (1718.02 ± 656.49), (1150.83 ± 371.53), and (541.52 ± 403.69) μg·min/mL. Within the tested dose range from 1.25 to 5.0 mL/kg, the values of t1/2 for the three major bioactive components were not affected by the dose, and AUC0-6h for BC and UDCA showed a good linearity to the dosage (r > 0.99), but AUC0-6h of CDCA was positively correlated with the dosage. After multiple iv administration of Tanreqing Injection (5.0 mL/kg), the values of AUC0-6h for BC, UDCA, and CDCA were (2872.37 ± 476.45), (3339.63 ± 939.94), and (1241.29 ± 408.38) μg·min/mL, and the accumulation index values were 83.3%, 118.5%, and 168.0%, respectively. Conclusion The elimination of BC and UDCA after single iv administration (1.25-5.0 mL/kg) is linear, and CDCA is probably linear after the administration. There is no significant accumulation in blood concentration for BC and UDCA, but CDCA has a tendency of accumulation after the multiple injection.

Progress in molecular mechanisms of HBV reverse transcription / 病毒学报

HBV infections leads to severe public health problems around the world, especially in China. Improved understanding of the molecular mechanisms of HBV reverse transcription is fundamental for optimization of treatment and solution to drug-resistance. Recently, the main structural basis involved in the process of HBV reverse transcription and the cis-elements were revealed by means of biochemistry and genetics. The entire process of reverse transcription is completed mainly through the first template switch mediated by the P- epsilon structure; the second template switch mediated by 5E/3E and M structure; and the third template switch mediated by 5' r / 3' r structure. The important structure and the cis-elements involved in this process are the focus of this review, at the same time, an overview of the progress in relevent studies is demonstrated to show the whole picture of the HBV reverse process.

Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system.</p><p><b>METHOD</b>26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively.</p><p><b>RESULT</b>Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test.</p><p><b>CONCLUSION</b>Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.</p>

Down-regulation of hepatitis B virus replication by heparin sulfate-D-glucosaminyl-3-O-sulfotransferase 3B1 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication.</p><p><b>METHODS</b>HepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference.</p><p><b>RESULT</b>Southern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity.</p><p><b>CONCLUSION</b>HS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.</p>

Detection of HBV resistant mutations related to lamivudine, adefovir and entecavir by reverse hybridization technique / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues.</p><p><b>METHODS</b>According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively.</p><p><b>RESULTS</b>The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing.</p><p><b>CONCLUSION</b>Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.</p>