scholarly journals Rebound of Hepatitis B Virus Replication in HepG2 Cells after Cessation of Antiviral Treatment

2002 ◽  
Vol 76 (16) ◽  
pp. 8148-8160 ◽  
Author(s):  
Ayman M. Abdelhamed ◽  
Colleen M. Kelley ◽  
Thomas G. Miller ◽  
Phillip A. Furman ◽  
Harriet C. Isom
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Drug Treatment ◽  
Time Course ◽  
Antiviral Treatment ◽  
Recombinant Baculovirus ◽  
Antiviral Agent ◽  
Hbv Dna ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Treatment of patients with lamivudine (3TC) results in loss of detectable levels of hepatitis B virus (HBV) DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated. Although the rebound observed in patients has also been seen in animal hepadnavirus models, rebound has not been analyzed in an in vitro cell culture system. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral agent-mediated loss of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure secreted virions, intracellular replicative intermediates, and nuclear non-protein-bound HBV DNA and separately analyze individual species of DNA, such as single-stranded HBV DNA compared to the double-stranded form and relaxed circular compared to covalently closed circular HBV DNA. We first determined that HBV replication in the HBV recombinant baculovirus/HepG2 system could proceed for at least 35 days, with a 30-day plateau level of replication, making it possible to study antiviral agent-mediated loss of HBV followed by rebound after cessation of drug treatment. All HBV DNA species decreased in a time-dependent fashion following antiviral treatment, but the magnitude of decline differed for each HBV DNA species, with the covalently closed circular form of HBV DNA being the most resistant to drug therapy. When drug treatment ceased, HBV DNA species reappeared with a pattern that recapitulated the initiation of replication, but with a different time course.

scholarly journals In Vitro Study of the Effects of Precore and Lamivudine-Resistant Mutations on Hepatitis B Virus Replication

2007 ◽  
Vol 81 (7) ◽  
pp. 3068-3076 ◽  
Author(s):  
Richard A. Heipertz ◽  
Thomas G. Miller ◽  
Colleen M. Kelley ◽  
William E. Delaney ◽  
Stephen A. Locarnini ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Time Course ◽  
Recombinant Baculovirus ◽  
Hbv Dna ◽  
Late Time ◽  
Time Points ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Understanding the consequences of mutation in the hepatitis B virus (HBV) genome on HBV replication is critical for treating chronic HBV infection. In this study, HBV replication in HepG2 cells initiated by transduction with precore (PC), rtM204I, and wild-type (wt) HBV recombinant baculoviruses was compared. The pattern and magnitude of HBV replication initiated by the PC HBV recombinant baculovirus were similar to those observed for wt HBV throughout the time course examined. In contrast, when the rtM204I mutation was introduced into wt HBV, by day 10 postinfection the levels of intra- and extracellular HBV DNA were markedly reduced compared to those for wt HBV. Although the rtM204I mutation reduced the production of HBV replicative intermediates, no effect on the level of covalently closed circular DNA or HBV transcripts was observed at late time points. Coinfection studies with different ratios of wt and rtM204I baculoviruses showed that the rtM204I variant did not produce a product that inhibited HBV replication. However, the combination of the wt and rtM204I baculoviruses yielded HBV DNA levels at late time points that were greater than those for the wt alone, suggesting that wt polymerase may function in trans to boost rtM204I replication. We concluded that the rtM204I mutation generates a polymerase that is not only resistant to lamivudine but also replicates nucleic acids to lower levels in vitro.

scholarly journals Penciclovir is a selective inhibitor of hepatitis B virus replication in cultured human hepatoblastoma cells.

1996 ◽  
Vol 40 (5) ◽  
pp. 1282-1284 ◽  
Author(s):  
B E Korba ◽  
M R Boyd
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Agent ◽  
Hbv Dna ◽  
Selective Inhibitor ◽  
Hbv Replication ◽  
Virion Release ◽  
B Virus ◽  
Immunodeficiency Virus ◽  
Untreated Culture

Penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yI)guanine], an effective antiherpesvirus agent, was found to be a potent and selective antiviral agent against intracellular hepatitis B virus (HBV) replication (drug concentration at which a 10-fold decrease in HBV DNA from the average level in an untreated culture was observed [EC90], 1.6 microM) and extracellular virion release (EC90, 0.7 microM) by cultured human hepatoblastoma (2.2.15) cells. Acyclovir and three other related 9-alkoxypurines with activity against either herpesviruses or human immunodeficiency virus were uniformly inactive against HBV. The activity of penciclovir is discussed in relation to recent findings related to its mode of action against HBV.

scholarly journals Comparison of Anti-Hepatitis B Virus Activities of Lamivudine and Clevudine by a Quantitative Assay

2003 ◽  
Vol 47 (1) ◽  
pp. 324-336 ◽  
Author(s):  
Ayman M. Abdelhamed ◽  
Colleen M. Kelley ◽  
Thomas G. Miller ◽  
Phillip A. Furman ◽  
Edward E. Cable ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Treatment ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Extracellular Dna ◽  
Quantitative Assay ◽  
Hbv Replication ◽  
B Virus

ABSTRACT In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC50) and EC90 values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.

scholarly journals Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

2004 ◽  
Vol 48 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Radhakrishnan P. Iyer ◽  
Yi Jin ◽  
Arlene Roland ◽  
John D. Morrey ◽  
Samir Mounir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Parallel Synthesis ◽  
Hbv Replication ◽  
Long Term Treatment ◽  
B Virus ◽  
Dna Strand

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

scholarly journals Automated Quantitative Analysis of Hepatitis B Virus DNA by Using the Cobas Amplicor HBV Monitor Test

1999 ◽  
Vol 37 (9) ◽  
pp. 2793-2797 ◽  
Author(s):  
Ulrika Noborg ◽  
Annkatrin Gusdal ◽  
Eva K. Pisa ◽  
Anders Hedrum ◽  
Magnus Lindh
Keyword(s):  
Quantitative Analysis ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Treatment ◽  
Colorimetric Detection ◽  
Hbv Dna ◽  
Linear Range ◽  
Virus Concentration ◽  
B Virus ◽  
The Mean

A highly sensitive method of quantitative analysis of hepatitis B virus (HBV) DNA in serum, the Cobas Amplicor HBV Monitor (Cobas-AM) test, was evaluated. Following a manual extraction of viral DNA, amplification, colorimetric detection, and quantitative determination are all automatically performed in the Cobas analyzer. Serially diluted samples with known HBV DNA concentrations were analyzed blindly. All samples with a virus concentration of 400 copies/ml and 83% of samples with a virus concentration of 100 copies/ml could be detected. A linear correlation between input HBV DNA and measured HBV DNA was seen in the range from 100 to 105 copies/ml. The mean coefficient of variation was 29.6% for all input levels and 18.9% for HBV DNA concentrations above 400 copies/ml. Samples with an HBV DNA level above 109 copies/ml could be reproducibly measured after predilution to 10−4 or 10−6 in negative serum; however, the level was underestimated if target DNA after dilution was still above the linear range of the assay. Quantitative results of the Cobas-AM test were interchangeable with measurements by the manual microwell plate version of Amplicor HBV Monitor (MWP-AM); the mean ratio for log Cobas-AM results/log MWP-AM results was 0.97 (standard error of the mean, 0.007) when serum samples from 153 chronic carriers were analyzed. The test should be of value for clinical assessment of chronic carriers and for monitoring the response to antiviral treatment. A limitation is the relatively narrow linear range of the assay, requiring predilution of high-titer (mainly hepatitis B e-antigen-positive) samples.

scholarly journals Hepatocyte Endoplasmic Reticulum Stress Inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance After Entecavir Treatment

2021 ◽  
Vol 7 ◽  
Author(s):  
Huan Chen ◽  
Maoyuan Mu ◽  
Qichuan Liu ◽  
Han Hu ◽  
Caiyun Tian ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Er Stress ◽  
Western Blotting ◽  
Hbv Dna ◽  
Translation Initiation Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hbv Replication ◽  
B Virus

Background: The aim of this study was to explore the effects of endoplasmic reticulum (ER) stress on hepatitis B virus (HBV) replication and the antiviral effect of entecavir (ETV).Methods: Thapsigargin (TG) and stearic acid (SA) were used to induce ER stress in HepG2.2.15 cells and HepAD38 cells that contained an integrated HBV genome, while ETV was used to inhibit HBV replication. The expression levels of glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic translation initiation factor 2 subunit alpha (p-eIF2α) were measured by western blotting. Intracellular HBV DNA was determined by qPCR; HBsAg by western blotting; HBV RNA by real-time RT-qPCR; HBsAg and HBeAg in supernatants by enzyme-linked immunosorbent assay (ELISA); and HBV DNA in supernatants by qPCR.Results: TG and SA induced ER stress in HepG2.2.15 cells and HepAD38 cells from 12 to 48 h post treatment. However, 4-phenylbutyric acid (PBA) partly alleviated the TG-induced ER stress. Moreover, TG inhibited HBsAg, HBeAg, and HBV DNA secretion from 12 to 48 h, while different concentrations of SA inhibited HBsAg and HBV DNA secretion at 48 h. TG promoted intracellular HBV DNA and HBsAg accumulation and the transcription of the HBV 3.5-kb mRNA and S mRNA. PBA treatment restored the secretion of HBsAg and HBV DNA. Finally, ER stress accelerated extracellular HBV DNA clearance but delayed intracellular HBV DNA clearance after ETV treatment.Conclusions: Hepatocyte ER stress promoted intracellular HBV DNA and HBsAg accumulation by inhibiting their secretion. Our study also suggested that hepatocyte ER stress delayed intracellular HBV DNA clearance after ETV treatment.

scholarly journals Strong and Selective Inhibitors of Hepatitis B Virus Replication among Novel N4-Hydroxy- and 5-Methyl-β-l-Deoxycytidine Analogues

2007 ◽  
Vol 51 (7) ◽  
pp. 2523-2530 ◽  
Author(s):  
E. Matthes ◽  
A. Funk ◽  
I. Krahn ◽  
K. Gaertner ◽  
M. von Janta-Lipinski ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Inhibitory Concentration ◽  
Hbv Dna ◽  
Selective Inhibitor ◽  
Effective Concentration ◽  
Active Inhibitor ◽  
Hbv Replication ◽  
B Virus

ABSTRACT Novel N4-hydroxy- and 5-methyl-modified β-l-deoxycytidine analogues were synthesized and evaluated as anti-hepatitis B virus (HBV) agents. Their in vitro efficiencies were investigated in HepG2.2.15 cells stably transfected with HBV. β-l-2′,3′-Didehydro-2′,3′-dideoxy-N4-hydroxycytidine (β-l-Hyd4C) was most effective in reducing secreted HBV DNA (50% effective concentration [EC50], 0.03 μM), followed by β-l-2′,3′-dideoxy-3′-thia-N4-hydroxycytidine (EC50, 0.51 μM), β-l-2′,3′-dideoxy-N4-hydroxycytidine (EC50, 0.55 μM), and β-l-5-methyl-2′-deoxycytidine (EC50, 0.9 μM). The inhibition of the presumed target, the HBV DNA polymerase, by the triphosphates of some of the β-l-cytidine derivatives was also assessed. In accordance with the cell culture data, β-l-Hyd4C triphosphate was the most active inhibitor, with a 50% inhibitory concentration of 0.21 μM. The cytotoxicities of some of the 4-NHOH-modified β-l-nucleosides were dramatically lower than those of the corresponding cytidine analogues with the unmodified 4-NH2 group. The 50% cytotoxic concentrations for β-l-Hyd4C in HepG2 and HL-60 cells were 2,500 μM and 3,500 μM, respectively. In summary, our results demonstrate that at least β-l-Hyd4C can be recommended as a highly efficient and extremely selective inhibitor of HBV replication for further investigations.

scholarly journals Experimental transfection of Macaca sylvanus with cloned human hepatitis B virus

2002 ◽  
Vol 83 (7) ◽  
pp. 1645-1649 ◽  
Author(s):  
Tarik Gheit ◽  
Souad Sekkat ◽  
Lucyna Cova ◽  
Michèle Chevallier ◽  
Marie Anne Petit ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Macaca Sylvanus ◽  
Primate Model ◽  
Virus Particles ◽  
Hbv Replication ◽  
Post Transfection ◽  
B Virus ◽  
Dane Particles

Due to the absence of easily accessible animal models for the study of hepatitis B virus (HBV), the possibility of using Macaca sylvanus, a monkey originating from Morocco, North Africa, was investigated. Three monkeys were intrahepatically inoculated with a replication-competent head-to-tail HBV DNA plasmid dimer construct. The HBV surface antigen and HBV DNA were detected prior to alanine aminotransferase elevation in the serum of two of three HBV-inoculated monkeys at day 2 post-transfection and persisted for several weeks. This indicates that transfected animals developed markers of HBV infection. In addition, electron microscopy of the serum 3 weeks post-transfection showed the presence of virus particles whose shape and size were similar to complete 42 nm HBV Dane particles. Histological examination of liver tissues also revealed pathological changes not observed in uninfected controls, which strongly suggested acute hepatitis. HBV DNA was also detected by PCR in these monkey livers. Taken together, these results indicate that HBV can successfully replicate in this model and that M. sylvanus could be a potentially useful new primate model for the study of HBV replication.

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