Objective To establish a method for quantitative detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA ) in infected cells. Methods The transfected cell line HepG2.2.15 which can consistently produce Dane particles was maintained in DMEM containing 380 ?g/ml G418 and 10% fetal bovine serum. Cells in the exponential period were harvested from flasks, then intracellular HBV cccDNA was extracted from pellet containing 1?10~6 cells with mini plasmid extraction kit (QIAGEN).The extraction product was further purified by mung bean nuclease to remove HBV relaxed circular DNA possibly remained. HBV cccDNA was quantitatively detected by fluorescent PCR with selective primer set and Taqman MGB probe. Culture medium before exponential period, HBV DNA positive and negative serum samples from patients with chronic hepatitis B (mild) were amplified simultaneously to test the specificity of the fluorescent PCR method. Plasmids containing whole HBV genome were amplified with the same primer set and fluorescent probe to determine the sensitivity of the method. Results HBV cccDNA was detectable in HepG2.2.15, and the average quantity was 18 copies per cell approximately. No detectable fluorescent signal was observed when culture and serum samples were amplified. The detectable HBV cccDNA was as low as 10~3 copies per ml at least by this method. Conclusions This method is convenient, highly specific and highly sensitive. It can be utilized in the quantitative detection of intracellular HBV cccDNA as well as in the screening and evaluation of antiviral agents.

Objective To detect nucleos(t)ide-resistance mutations in hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) isolated from hepatocytes of patients with chronic HBV infection and to analyze the correlation between the mutations found in cccDNA and relaxed circular DNA (rcDNA). MethodsForty patients with chronic HBV infection were investigated. Preoperation serum samples and non-tumor liver tissues were collected.Intrahepatic HBV cccDNA and rcDNA were selectively extracted by co-precipitation of sodium dodecyl sulphate-protein and QIAamp DNA Mini Kit, and further purification with plasmid-safe ATP-dependent DNase (PSAD).Thereafter,cccDNA were amplified by selective polymerase chain reaction (PCR) or nested PCR using the primers spanning both the two gaps in HBV genome and covering the common mutations associated with nucleoside analogues resistance (rt169- rt250).Intrahepatic HBV rcDNA and pre-operation serum HBV rcDNA were also extracted and then amplified by PCR.The PCR products were then purified and sequenced.Results Among the 40 patients,intrahepatic HBV cccDNA were detected in 31 patients,and HBV rcDNA were detected in liver samples of 35 patients and pre-operation serum samples of 21 patients. The PCR products amplified from these samples were all successfully sequenced.rtM204Ⅰ mutation was detected in intracellular HBV cccDNA,rcDNA and serum HBV rcDNA in 2 patients.Both rtM204Ⅰ and rtQ215H were detected in intrahepatic HBV cccDNA and rcDNA in 2 patients,while no corresponding mutation was observed in serum HBV rcDNA of these two patients.Three variants including rtM204V,rtM204V and rtV173L-rtL180M-rtM204V were detected in serum HBV rcDNA in 3 patients,while corresponding mutants were not detected in intracellular HBV cccDNA or rcDNA of these patients.Condsions The results suggest that antiviral nucleos(t) ide resistance mutations can be found in HBV cccDNA in chronic hepatitis B patients. The dominant resistant mutation found in intrahepatic HBV cccDNA/rcDNA may be different from that in serum HBV rcDNA.

Objective To determine whether hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) could be detected in serum of patients with hepatitis B and evaluate the related factors and clinical significances. Methods Fifty-seven patients, including 26 with mild chronic hepatitis B (CHB) and 31 with severe hepatitis B (SHB) were enrolled. Prothrombin time (PT), hepatic biochemical indexes, serum markers of hepatitis virus, serum total HBV DNA and HBV cccDNA of every patient were detected after hospitalization. Factors associated with the detection rate of serum HBV cccDNA were analyzed using Logistic stepwise regression. Results Serum HBV cccDNA was detected in 13 patients with SHB and 1 with mild CHB, and serum levels of HBV cccDNA were varying from 1.25 × 103 to 4. 88 × 104 copy/mL. The detection rates were significantly different between the two groups (P=0. 0014). The sensitivity and specificity of SHB diagnose by serum HBV cccDNA detection were 41.94% and 96.15 %, respectively. Logistic regression analysis showed that the detection rate of serum HBV cccDNA was associated with PT (X2 = 7. 2192, P= 0. 0072), while not associated with age, sex, total serum HBV DNA, total bilirubin or alanine aminotransferase (ALT). Conclusion Serum HBV cccDNA could be detected in some of the patients with SHB, whic hmay be considered as one of the diagnostic indexes for SHB,

Anti-Bacterial Agents ; adverse effects ; Anti-Infective Agents ; adverse effects ; Anti-Inflammatory Agents ; adverse effects ; Biomarkers ; blood ; Chemical and Drug Induced Liver Injury ; diagnosis ; epidemiology ; Child ; Drug-Related Side Effects and Adverse Reactions ; Humans ; Liver ; drug effects ; metabolism ; pathology ; Pediatrics ; Risk Factors

Objective To determine the effect of oxymatrine on the activity of HBV DNA polymerase in vitro. Methods Hepatitis B virus particles were purified from supernatant of cultured HepG2.2.15 cells by uhracentrifugation, and then were mixed with reaction buffer containing NP-40, β-mercaptoethanol, 32P-labelled nucleoside triphosphate (dCTP), MgCl2, and different concentrations of oxymatrine ( 1000 μg/ml, 800 μg/ml, 600 μg/ml, 400 μg/ml and 200 μg/ml) or adefovir dipivoxil ( 100 μg/ml, 80 μg/ml and 60 μg/ml, 40 μg/ml and 20 μg/ml). After incubation at 37 ℃ overnight, proteinase K was added to the reaction system for digestion and 35 μl of samples were spotted onto DE81 paper. Activities of endogenous polymerase in HBV particles were assessed by determining the radioactivity of 32P-labelled dCTP incorporated in the plus-strain of viral DNA. Results Compared with the blank control, the activity of endogenous polymerase in HBV particles treated with different doses of oxymatrine varied from 103% to 107%, and it varied from 91% to 101% when treated with different doses of adefovir dipivoxil. No significant difference was observed among treated groups and the control (P > 0. 05 ). Conclusion No direct inhibitory effect of oxymatrine on the activity of HBV polymerase was observed in vitro.

Objective To identify hepatitis B virus X-interactive proteins by comparative proteomics method and to understand the molecular mechanism of HBx in the development of hepatocellular carcinoma(HCC).Methods Two-dimensional gel electrophoresis(2-DE)was used to separate the total proteins of HBx-transfected human hepatoma cell lines HepG2-Px and its parental cell lines HepG2-P_0.PDQuest software was applied to analyze 2 DE images.The differentially expressed protein spots between the two cell lines were identified by matrix-assisted laser desorptionionization time of flight mass spectrometry(MALDI-TOF-MS)and electrospray ionization tandem mass spectrometry(ESI-Q-TOF).Then,the differential expression levels of some identified proteins were determined by Western blot.The data were compared using t test.Results The well-resolved,reproducible 2-DE patterns of HepG2-Px and HepG2-P_0 total proteins were established.A total of 32 differential proteins were identified in HepG2-Px cell,including 25 up-regulated proteins,such as heat shock protein(HSP)90AB1,Bcl-2 associated athanogene(BAG)-2,nucleophosmin(B23),chloride intracellular channel(CLIC)-1,matrix metalloproteinase(MMP)-3,melanoma antigen(MAGE)-12,and 7 down-regulated proteins,such as Wnt-5a.The differential expression levels of some proteins between the two cell lines were confirmed by Western blot analysis.Conclusions Most of the identified proteins are involved in many processes,such as transcription,signal transduction,cell proliferation,cell cycle regulator,apoptosis,DNA repair,metabolisms and immunity.These differential proteins may play a role in tumor genesis and HC development.The data are valuable for further study on the role of HBx in human hepatocellular carcinoma.

Ohjective To observe the inhibition effect of total glucosides of Picrorhiza on hepatitis B virus covalently closed circular DNA (HBV cccDNA) in HepG 2.2.15 cell line. Methods HepG 2.2.15 cells were incubated with culture medium containing 50 mg/L of picrosides or 5 mg/L of adefovir dipivoxil for 2 or 5 days. HBV DNA in the supernatant, intracellular cccDNA, relaxed circular DNA (rcDNA) and pregenomic RNA (pgRNA) were quantified by specific real-time polymerase chain reaction (RT-PCR) and inhibition rates were calculated. The means were compared by t test. Results After treated with picrosides for 2 and 5 days, the inhibition rates of HBV DNA in thesupernatant were 49. 74% (t=4.723, P<0.05) and 79.48% (t = 7.512, P<0.05), respectively. The inhibition rates of intracellular cccDNA were 43.55% (t = 5.216, P<0.05) and 56.43% (t=7.262, P<0.05), respectively, while those of intracellular rcDNA were 43.39% (t=4.137, P<0.05) and 63.86% (t=7.861, P<0.05), respectively, and those of intracellular pgRNA were 54.72% (t=4.532, P<0.05) and 56.08% (t=4.833, P<0.05), respectively. Comparatively, after treatment with adefovir dipivoxil for 2 and 5 days, the inhibition rates of HBV DNA in the supernatant were 25.56% (t=2.874, P<0.05) and 92.44% (t =10.276, P<0.05), respectively. Those of cccDNA were 18.54% (t=2.736, P<0.05) and 47.19% (t=6.852, P<0.05), respectively. Those of rcDNA were 21. 20% (t=3.206, P<0.05) and 71.47% (t=8.332, P<0.05), respectively, pgRNA were 11.14% (t=1.761, P>0.05) and 37.61%(t=3.632, P<0.05) respectively in HepG2.2.15 cells. Conclusions Pierosides may inhibit the replication cycle of HBV, including the formation of cccDNA in HepG 2.2.15 cells. The mechanism of pierosides on cccDNA may differ from adefovir dipivoxil's due to its earlier inhibition time phase.

Objective:To investigate the changes of liver pathology and its influencing factors in patients with chronic hepatitis B viral (HBV) infection and low level of serum alanine aminotransferase (ALT).Methods:The clinical data of 135 with chronic HBV infection patients, in whom the serum ALT levels were less than two times of the upper limit of normal (ULN), were collected from the Affiliated Hospital of Medical School of Ningbo University and the First Affiliated Hospital, Zhejiang University School of Medicine during July 2017 and July 2019. The result of hepatic histological examination was reviewed, and the risk factors of obvious liver inflammation (G≥2) or fibrosis (S≥2) in patients were analyzed with Logistic regression analysis.Results:The pathological examination of liver tissue revealed G≥2 or S≥2 in 52 cases (38.5%). The univariate analysis showed that age, family history of HBV infection, ALT 1-<2×ULN, aspartate aminotransferase(AST)≥1×ULN, low platelet count(PLT)and prolongation of prothrombin time(PT)were associated with G≥2 or S≥2 in chronic HBV infection patients with low level ALT ( P<0.05 or <0.01). Multivariate Logistic regression analysis showed that age( OR=1.052, 95% CI 1.007-1.100), family history of HBV infection( OR=5.448, 95% CI 2.191-13.548)and AST( OR=1.042, 95% CI 1.005-1.081)were independent risk factors of G≥2 or S≥2 in chronic HBV infection patients with low level ALT ( P<0.05 or <0.01). Conclusion:Age, family history of HBV infection and AST level can be used to judge the severity of liver pathological changes and necessity of antiviral treatment for patients with chronic HBV infection having ALT<2×ULN.

Objective:To investigate the incidence of liver injury in patients with coronavirus disease 2019 (COVID-19), and to explore its impact on the condition and prognosis of patients.Methods:The medical records of 67 patients with COVID-19 who presented with pneumonia hospitalized at Tongji Hospital, Huazhong University of Science and Technology from February 11 to March 28, 2020 were collected. The results of liver biochemistry and coagulation function test at admission were analyzed. Data were compared by chi-square test, analysis of variance or Kruskal-Wallis H test. Results:Among 67 patients, total bilirubin increased in seven (10.4%) patients, which was slightly abnormal, albumin decreased in 36(53.7%) cases, and was below 30 g/L in 15(22.4%) cases, alanine transaminase (ALT) and aspartate transaminase (AST) were elevated in 19(28.4%) and 12(17.9%) cases, respectively. A total of 22(32.8%) cases had elevated ALT and (or) AST. The incidences with elevated ALT and (or) AST in moderate and severe patients were 33.3%(10/30) and 26.9%(7/26), respectively. Five of 11 critical patients had elevated ALT and (or) AST. There was no significant difference among the three groups ( χ2=1.21, P=0.546). Abnormal alkaline phosphatase and (or) γ-glutamyl transpeptidase were observed in 11(16.4%) cases. The prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) occurred in 10(14.9%) and 17(25.4%) patients, respectively, while most of them were slightly abnormal. Only one patient presented with prolongation of PT and APTT meeting the standard of liver failure. A total of 61.2%(41/67) and 65.7%(44/67) of cases showed increase of fibrinogen and D-dimer, respectively, and 28.4%(19/67) and 19.4%(13/67), respectively increased to an obvious extent. The albumin levels in moderate, severe and critical patients were (37.85±6.19) g/L, (32.96±4.33) g/L and (33.02±3.63) g/L, respectively, which were significantly different ( F=7.36, P=0.001). There were significant differences in PT, APTT, fibrinogen and D-dimer among the three groups ( F=3.22, 3.31, 4.06 and H=17.63, respectively, all P<0.05). Conclusions:COVID-19 only leads to mild liver injury and has only mild impact on liver function. The decrease of albumin level and the increase of fibrinogen and D-dimer may be early predicting indexes for the disease severity.

Objective:To reevaluate the upper limit of normal (ULN) of serum alanine aminotransferase (ALT) by retrospectively analyzing the ALT levels in healthy people in Ningbo area.Methods:A total of 56 140 people who underwent health examination and detection of liver biochemical indexes in the Affiliated Hospital of Medical School of Ningbo University and Yinzhou Huamao Hospital of Ningbo from 2018 to 2020 were enrolled. After excluding relevant factors that may lead to liver injury, 11 411 people were included to compare the difference of serum ALT levels among different genders and age groups (20 to 29 years, 30 to 39 years, 40 to 49 years and 50 to 59 years) to determine the ALT ULN in different gender groups. Statistical methods were performed using two independent samples t test and analysis of variance. Results:The serum ALT of males was (19.20±7.90) U/L, which was higher than that of females ((13.75±6.17) U/L), with statistical significance ( t=41.16, P<0.001). The serum ALT ULN in males and in females were 35 U/L and 26 U/L, respectively. The serum ALT levels of 20 to 29, 30 to 39, 40 to 49 and 50 to 59 years old groups were (15.48±7.61) U/L, (16.21±7.40) U/L, (17.36±7.52) U/L and (18.77±7.57) U/L, respectively.The difference was statistically significant ( F=71.51, P<0.001). Serum ALT level in 50 to 59 years old group was higher than that in 20 to 29 years old group, and the difference was statistically significant ( t=13.11, P<0.01). In males, the ALT ULN of 20 to 29 years old was the lowest of 34.43 U/L, and highest of 35.29 U/L in 40 to 49 years old. In females, the ALT ULN in the 20 to 29 years old group was the lowest of 23.01 U/L, and the ALT ULN in the 50 to 59 years old group was the highest of 30.79 U/L. ALT ULN increased with age in females. The serum ALT of males was higher than that of females in all age groups ( t=29.55, 26.91, 13.43 and 4.62, respectively, all P<0.05). Conclusions:The serum ALT level is significantly correlated to gender and age. The serum ALT ULNs of healthy adult are 35 U/L in males and 26 U/L in females in Ningbo area.