Classical drug resistance mutations of HepG2.2.15 cells persistently induced by appropriate concentrations of adefovir / 解放军医学杂志

Objective To observe whether the classic drug-resistant mutations can be induced in various concentrations of adefovir (ADV)-treated HepG2.2.15 cells persistently and explore the mechanism for emergence of drug resistance.Methods HepG2.2.15 cells were cultured continually in 12-well plates with medium containing 0,0.01,0.1,1.0μmol/L concentration of ADV,and passaged every 3 days up to the 110th generations.The intracellular and supernatant HBV DNA was extracted every 10 generations.Intracellular HBV cccDNA was amplified by plasmid-safe ATP dependent DNase (PSAD) digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR assay.And the RT region of supernatant HBV DNA was amplified by one-tube nested PCR assay.Then the classic drug-resistant mutations of the RT region of intracellular cccDNA and supernatant HBV DNA were analyzed using direct PCR sequencing combined with clonal sequencing (more than 20 clones/sample).Results HBV DNA stably replicated in ADV-untreated cells (control group).The intracellular total DNA and cccDNA levels,supernatant HBV DNA level decreased continuously with the prolonged ADV culture duration in 0.01 μmol/L and 0.1μmol/L ADV group.Drug resistant mutations were not detected up to the 110th generation in 0.01 μmol/L ADV group;while rtA181V+N236T mutations were detected at the110th generation in 0.1μmol/L ADV group.The 1.0μmol/L ADV group was ceased of culture at the 15th generation due to inhibited cell growth.Conclusion HBV cccDNA exists in HepG2.2.15 cells,and the classical drug-resistant mutations of rtA181V+N236T could be induced by proper concentration of ADV.

The clinical efficacy and safety of adefovir dipivoxil in combination with bicyclol for the treatment of senior patients with chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the clinical efficacy and safety of adefovir dipivoxil (ADV) in combination with bicyclol for the treatment of chronic hepatitis B (CHB) in seniors.</p><p><b>METHODS</b>96 senior patients with CHB were randomly divided into two groups, the treatment group and the control group. On the basis of routine liver protective treatment, patients in the treatment group received ADV (10 mg/d) and bicyclol tablets (25 mg, tid.) orally, and those in the control group were orally administrated ADV tablets (10 mg/d) only. The treatment course for both groups was 24 weeks. Serum ALT, AST, and alterations of virological parameters were observed before and after the treatment.</p><p><b>RESULTS</b>Before and at the end of the 24 weeks treatment, ALT level for the treatment group was (208.44 +/- 94.22) and (34.47 +/- 12.79) U/L, and those for the control group was (205.73 +/- 96.48) and (44.20 +/- 21.96) U/L, respectively (difference between groups P < 0.01). At the end of the 24 weeks treatment, ALT normalization rates for the treatment group and the control group were 76.6% and 54.5%, respectively, and AST normalization rates for them were 76.6% and 54.5%, respectively (both differences between groups P < 0.05); HBV DNA loads for the treatment group and the control group were decreased by (3.1 +/- 1.40) lgIU/ml and (2.98 +/- 1.17) lgIU/ ml, respectively (difference between groups P > 0.05). The incidence rates of adverse events between two groups were not statistically significant.</p><p><b>CONCLUSION</b>It suggested that the treatment of ADV in combination with bicyclol for senior patients with CHB is effective and safe.</p>

Construction and identification of attenuated Salmonella which harboring enterovirus 71 VP1 gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop attenuated Salmonella which harboring enterovirus 71 (EV71) VP1 gene.</p><p><b>METHODS</b>The plasmid which expressed VP1 protein of EV71 was constructed by gene recombination. Cellular expression was assessed by Western Blot analysis. The recombinant plasmid was then transformed into attenuated Salmonella SL7207.</p><p><b>RESULTS</b>EV71 VP1 gene sequence was inserted into a eukaryotic expression plasmid VR1012. VP1 protein was detected by Western Blot analysis in the culture supernatant. And the attenuated Salmonella harbored the plasmid stable.</p><p><b>CONCLUSION</b>The plasmid was constructed successfully and it can express effectively in vitro. The bacteria which harboring the plasmid were constructed successfully. This has provided a basis for further research of an oral EV71 vaccine.</p>

Clinical research on the effect of Oxymatrine on serum cholinesterase / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the effect of Oxymatrine (OM) on serum cholinesterase (ChE) during the treatment of viral hepatitis and the relationship between the change of ChE and the change of albumin (ALB), prothrombin activity (PTA) and other liver function tests.</p><p><b>METHODS</b>A total of 98 patients with viral hepatitis were divided into four groups. Group A consisted of 31 patients and were treated with OM intravenous infusion; Group B consisted of 30 patients, treated with OM orally; Group C consisted of 7 patients and were treated with OM intramuscular injection while Group D consisted of 30 patients, and were not treated with OM. ChE, ALB, PTA, liver function, renal function, soluble complement receptor-1 (sCR1) and erythrocyte innate immune adhesion function (EIIAF) were regularly determined.</p><p><b>RESULTS</b>ChE in Group A,B,C was dropped obviously during the treatment (P less than 0.001, less than 0.001, 0.023=. But there were no change in ALB, PTA, sCR1, EIIAF (P greater than 0.05), and remarkable improvement of ALT, AST, TBiL was seen during the treatment in Groups A, B, C. After the treatment with OM, the level of ChE recovered soon.</p><p><b>CONCLUSION</b>Serum level of ChE significantly declined during the treatment of viral hepatitis with OM, but no change was found in ALB, PTA, sCR1, EIIAF while liver function tests showed better results. So the drop of ChE does not mean deprivation of patient's liver disease.</p>