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1.
Virol Sin ; 33(6): 538-544, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30421112

RESUMO

2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 µmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovir-resistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.


Assuntos
Antivirais/farmacologia , Didesoxinucleosídeos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleosídeos/farmacologia , RNA Viral/efeitos dos fármacos
2.
Biomolecules ; 8(3)2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013006

RESUMO

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.


Assuntos
Antivirais/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Hepadnaviridae/fisiologia , Ácidos Nucleicos Peptídicos/administração & dosagem , Administração Intravenosa , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Modelos Animais de Doenças , Patos , Hepadnaviridae/efeitos dos fármacos , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B do Pato/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Humanos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Replicação Viral/efeitos dos fármacos
3.
Hepatology ; 67(6): 2127-2140, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29251788

RESUMO

Nucleic acid polymer (NAP) REP 2139 treatment was shown to block the release of viral surface antigen in duck HBV (DHBV)-infected ducks and in patients with chronic HBV or HBV/hepatitis D virus infection. In this preclinical study, a combination therapy consisting of REP 2139 with tenofovir disoproxil fumarate (TDF) and entecavir (ETV) was evaluated in vivo in the chronic DHBV infection model. DHBV-infected duck groups were treated as follows: normal saline (control); REP 2139 TDF; REP 2139 + TDF; and REP 2139 + TDF + ETV. After 4 weeks of treatment, all animals were followed for 8 weeks. Serum DHBsAg and anti-DHBsAg antibodies were monitored by enzyme-linked immunosorbent assay and viremia by qPCR. Total viral DNA and covalently closed circular DNA (cccDNA) were quantified in autopsy liver samples by qPCR. Intrahepatic DHBsAg was assessed at the end of follow-up by immunohistochemistry. On-treatment reduction of serum DHBsAg and viremia was more rapid when REP 2139 was combined with TDF or TDF and ETV, and, in contrast to TDF monotherapy, no viral rebound was observed after treatment cessation. Importantly, combination therapy resulted in a significant decrease in intrahepatic viral DNA (>3 log) and cccDNA (>2 log), which were tightly correlated with the clearance of DHBsAg in the liver. CONCLUSION: Synergistic antiviral effects were observed when REP 2139 was combined with TDF or TDF + ETV leading to control of infection in blood and liver, associated with intrahepatic viral surface antigen elimination that persisted after treatment withdrawal. Our findings suggest the potential of developing such combination therapy for treatment of chronically infected patients in the absence of pegylated interferon. (Hepatology 2018;67:2127-2140).


Assuntos
Antivirais/administração & dosagem , Guanina/análogos & derivados , Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Ácidos Nucleicos/administração & dosagem , Polímeros/administração & dosagem , Tenofovir/administração & dosagem , Animais , Doença Crônica , Sinergismo Farmacológico , Quimioterapia Combinada , Patos , Guanina/administração & dosagem
4.
Eur J Med Chem ; 143: 137-149, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29174810

RESUMO

Novel drugs are urgently needed to combat hepatitis B virus (HBV) infection due to drug-resistant virus. In this paper, a series of novel 4-monosubstituted 2'-deoxy-2'-ß-fluoro-4'-azido-ß-d-arabinofuranosyl 1,2,3-triazole nucleoside analogues (1a-g) were designed, synthesized and screened for in vitro anti-HBV activity. At 5.0 µM in the cellular model, all the synthetic compounds display activities comparable to that of the positive control, lamivudine at 20 µM. Of the compounds tested, the amide-substituted analogue (1a) shows the most promising anti-HBV activity and low cytotoxicity in the cell model. In particular, it retains excellent activity against lamivudine-resistant HBV mutants. In duck HBV (DHBV)-infected duck models, both the serum and liver DHBV DNA levels (67.4% and 53.3%, respectively) were reduced markedly by the treatment with 1a. Analysis of the structure of HBV polymer/1a-triphosphate (1a-TP) complex shows that 1a-TP is stabilized by specific van der Waals interactions with the enzyme residues arising from 4-amino-1,2,3-triazole and the 4'-azido group.


Assuntos
Antivirais/farmacologia , Desenho de Drogas , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Monossacarídeos/farmacologia , Nucleosídeos/farmacologia , Triazóis/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Patos , Células Hep G2 , Vírus da Hepatite B/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Monossacarídeos/síntese química , Monossacarídeos/química , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Replicação Viral/efeitos dos fármacos
5.
Mol Med Rep ; 16(5): 7199-7204, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28944845

RESUMO

Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR­associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model. We designed six single­guide RNAs (sgRNA1­6) targeting the DHBV genome. The sgRNA/Cas9 plasmid was transfected into DHBV­infected PDHs, and then DHBV total DNA (in culture medium and PDHs) and cccDNA were quantified by reverse transcription­quantitative polymerase chain reaction. The combined inhibition of CRISPR/Cas9 system and entecavir (ETV) was also assessed. Two sgRNAs, sgRNA4 and sgRNA6, exhibited efficient inhibition on DHBV total DNA (77.23 and 86.51%, respectively), cccDNA (75.67 and 85.34%, respectively) in PDHs, as well as DHBV total DNA in the culture medium (62.17 and 59.52%, respectively). The inhibition remained or enhanced from day 5 to day 9 following transfection. The combination of the CRISPR/Cas9 system and ETV further increased the inhibitory effect on DHBV total DNA in PDHs and culture medium, but not cccDNA. The CRISPR/Cas9 system has the potential to be a useful tool for the suppression of DHBV DNA.


Assuntos
Sistemas CRISPR-Cas/genética , DNA Viral/metabolismo , Vírus da Hepatite B do Pato/genética , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sequência de Bases , DNA Viral/análise , DNA Viral/antagonistas & inibidores , Patos , Edição de Genes , Guanina/análogos & derivados , Guanina/farmacologia , Guanina/uso terapêutico , Hepatite B/tratamento farmacológico , Hepatite B/veterinária , Hepatite B/virologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/química , RNA Guia/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência
6.
Antiviral Res ; 131: 40-8, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27083116

RESUMO

Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Hepatite B do Pato/genética , Vírus da Hepatite B/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Viral/metabolismo , Antivirais/farmacologia , Carcinoma Hepatocelular , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B do Pato/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Humanos , Interferon-alfa/farmacologia , Neoplasias Hepáticas , Nucleocapsídeo/metabolismo , RNA/metabolismo , RNA Viral/isolamento & purificação , Ribonuclease H/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
7.
Int J Mol Sci ; 16(12): 28230-41, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26633356

RESUMO

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.


Assuntos
Antivirais/administração & dosagem , Biopolímeros , Cátions , Peptídeos Penetradores de Células , Portadores de Fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/prevenção & controle , Animais , Biopolímeros/química , Cátions/química , Quitosana/química , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Humanos , Imunidade Celular , Imunidade Humoral , Ácidos Nucleicos Peptídicos/administração & dosagem , Replicação Viral/efeitos dos fármacos
8.
J Ethnopharmacol ; 160: 1-5, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25446633

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthus ilicifolius L. is an important medicinal mangrove plant. It is popularly used for its anti-inflammatory, antioxidant activity and hepatoprotective effects. The present study was conducted to evaluate the effect of treatment with alcohol extract of Acanthus ilicifolius L. on duck hepatitis B. MATERIALS AND METHODS: One-day-old Guangxi shelducks injected intraperitoneally with strong positive duck hepatitis B virus (DHBV) serum were used to establish a duck hepatitis B animal model in the study. The ducks were respectively administered in different groups with low-, middle- and high-dose alcohol extracts of Acanthus ilicifolius L., the positive control drug acyclovir (ACV) and double-distilled water. The levels of serum DHBV DNA were detected by fluorescence quantitative PCR (FQ-PCR). Duck hepatitis B surface antigen (DHBsAg) and duck hepatitis B e antigen (DHBeAg) OD values in the serum were measured by ELISA. The activity of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) in the serum was measured, and the livers were taken for histopathological examination. RESULTS: The levels of serum DHBV DNA and the values of DHBsAg and DHBeAg OD were not significant in any of the dose extract groups. However, the ALT activity was obviously lower in the middle- and high-dose extract groups. It was also found that a high dose of alcohol extract could reduce the activity of AST significantly and significantly improve hepatic pathological effects. CONCLUSIONS: High-dose alcohol extract of Acanthus ilicifolius L. has an obvious protective effect on the liver function and liver tissue. However, the present study finds that Acanthus ilicifolius L. cannot inhibit the replication of duck hepatitis B virus.


Assuntos
Acanthaceae/química , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Fígado/efeitos dos fármacos , Fitoterapia , Aciclovir/farmacologia , Aciclovir/uso terapêutico , Alanina Transaminase/sangue , Animais , Animais Recém-Nascidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Aspartato Aminotransferases/sangue , DNA Viral/sangue , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/química , Patos , Infecções por Hepadnaviridae/patologia , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite Viral Animal/patologia , Fígado/patologia , Carga Viral/efeitos dos fármacos
9.
J Pharmacol Sci ; 126(3): 208-15, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25409748

RESUMO

A novel codrug, α-DDB-FNCG, was synthesized through coupling of α-biphenyl dimethyl dicarboxylate (α-DDB) and the nucleoside analogue FNCG, via an ester bond. The anti-HBV activity and hepatoprotective effects of this compound were investigated both in vitro and in vivo. In HBV-transfected HepG2.2.15 cell line, the secretion of HBsAg and HBeAg as well as the levels of extracellular and intracellular viral DNA were determined by ELISA and real-time fluorescent quantitative Polymerase Chain Reaction (FQ-PCR), respectively. In DHBV-infected ducks, the viral DNA levels in serum and liver were determined by FQ-PCR. In addition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and liver were also examined. The improvement of ducks' livers was evaluated by histopathological analysis. It has been demonstrated that α-DDB-FNCG could suppress the levels of HBV antigens and viral DNA in a time- and dose-dependent manner in the HepG2.2.15 cell line. Furthermore, this codrug could also significantly inhibit the viral DNA replication and reduce the ALT and AST levels in both serum and liver of DHBV-infected ducks, with improved hepatocellular architecture in drug-treated ducks. In short, these results suggest that α-DDB-FNCG could be a promising candidate for further development of new anti-HBV agents with hepatoprotective effects.


Assuntos
Antivirais/farmacologia , Benzodioxóis/farmacologia , Citidina/análogos & derivados , Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Fígado/efeitos dos fármacos , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Citidina/farmacologia , DNA Viral/metabolismo , Relação Dose-Resposta a Droga , Patos , Feminino , Células Hep G2 , Infecções por Hepadnaviridae/sangue , Infecções por Hepadnaviridae/patologia , Infecções por Hepadnaviridae/virologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B do Pato/genética , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite Viral Animal/sangue , Hepatite Viral Animal/patologia , Hepatite Viral Animal/virologia , Humanos , Fígado/enzimologia , Fígado/patologia , Fígado/virologia , Masculino , Fatores de Tempo , Transfecção , Carga Viral
10.
J Ethnopharmacol ; 157: 62-8, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25260580

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Nirtetralin B, a new lignan first reported by our team, is isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of nirtetralin B using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models. MATERIALS AND METHODS: Nirtetralin B was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after nirtetralin B was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks. RESULTS: In the human HBV-transfected liver cell line HepG2.2.15, nirtetralin B effectively suppressed the secretion of the HBV antigens in a dose-dependent manner with IC50 values for HBsAg of 17.4µM, IC50 values for HBeAg of 63.9µM. In DHBV-infected ducklings, nirtetralin B significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. And analysis of the liver pathological changes confirmed the hepatoprotective effect of nirtetralin B. CONCLUSION: The experimental data demonstrated that nirtetralin B exhibits anti-hepatitis B virus activity both in vitro and in vivo.


Assuntos
Anisóis/farmacologia , Antivirais/farmacologia , Dioxóis/farmacologia , Hepatite B/tratamento farmacológico , Lignanas/farmacologia , Phyllanthus/química , Animais , Anisóis/administração & dosagem , Anisóis/isolamento & purificação , Antivirais/administração & dosagem , Antivirais/isolamento & purificação , Dioxóis/administração & dosagem , Dioxóis/isolamento & purificação , Relação Dose-Resposta a Droga , Patos , Feminino , Células Hep G2 , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B do Pato/efeitos dos fármacos , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Lignanas/administração & dosagem , Lignanas/isolamento & purificação , Masculino , Medicina Tradicional , Replicação Viral/efeitos dos fármacos
11.
J Ethnopharmacol ; 155(2): 1061-7, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25009077

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Niranthin is a lignan isolated from Phyllanthus niruri L. This plant has long been used in folk medicine for liver protection and antihepatitis B in many Asian countries. This study was designed to evaluate the anti-hepatitis B virus activity of niranthin using HepG2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models. MATERIALS AND METHODS: Niranthin was isolated from Phyllanthus niruri L. (Euphorbiaceae) by extraction and chromatographic procedures and the anti-hepatitis B virus activity was evaluated both in vitro and in vivo. The human HBV-transfected liver cell line HepG2.2.15 was used in vitro assay. And the in vivo anti-hepatitis B virus activity was evaluated on the expression of HBV replication, HBsAg, HBeAg, ALT and AST on day 0, 7, 14, 17 after niranthin was dosed intragastricly (i.g.) once a day for 14 days at the dosages of 25, 50 and 100 mg/kg/day in the duck hepatitis B virus (DHBV) infected ducks. RESULTS: In the human HBV-transfected liver cell line HepG2.2.15, the secretion of HBsAg and HBeAg were significantly decreased after treatment with niranthin for 144 h, with IC50 values for HBsAg of 15.6 µM, IC50 values for HBeAg of 25.1 µM. In DHBV-infected ducklings, niranthin significantly reduced the serum DHBV DNA, HBsAg, HBeAg, ALT and AST. Furthermore, analysis of the liver pathological changes confirmed the hepatoprotective effect of niranthin. CONCLUSION: The experimental data demonstrated that niranthin exhibits anti-hepatitis B virus activity both in vitro and in vivo.


Assuntos
Anisóis/farmacologia , Antivirais/farmacologia , Dioxóis/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Phyllanthus/química , Animais , Anisóis/administração & dosagem , Anisóis/isolamento & purificação , Antivirais/administração & dosagem , Antivirais/isolamento & purificação , Dioxóis/administração & dosagem , Dioxóis/isolamento & purificação , Modelos Animais de Doenças , Patos , Feminino , Células Hep G2 , Infecções por Hepadnaviridae/tratamento farmacológico , Infecções por Hepadnaviridae/virologia , Hepatite B/tratamento farmacológico , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B do Pato/efeitos dos fármacos , Antígenos E da Hepatite B/metabolismo , Hepatite Viral Animal/tratamento farmacológico , Hepatite Viral Animal/virologia , Humanos , Concentração Inibidora 50 , Lignanas/administração & dosagem , Lignanas/isolamento & purificação , Lignanas/farmacologia , Masculino
12.
Biol Pharm Bull ; 37(7): 1214-20, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24759764

RESUMO

Hepatitis B is the most common serious liver infection in the world. To date, there is still no complete cure for chronic hepatitis B. Natural caffeic acid analogues possess prominent antiviral activity, especially anti-hepatitis B virus (HBV) and anti-human immunodeficiency virus effects. Cichoric acid is a caffeic acid derivative from Cichorium intybus. In the study, the anti-hepatitis B property of cichoric acid was evaluated by the D-galactosamine (D-GalN)-induced normal human HL-7702 hepatocyte injury model, the duck hepatitis B virus (DHBV)-infected duck fetal hepatocytes and the HBV-transfected cell line HepG2.2.15 cells, respectively. The results showed that cichoric acid attenuated significantly D-GalN-induced HL-7702 hepatocyte injury at 10-100 µg/mL and produced a maximum protection rate of 56.26%. Moreover, cichoric acid at 1-100 µg/mL inhibited markedly DHBV DNA replication in infected duck fetal hepatocytes. Also, cichoric acid at 10-100 µg/mL reduced significantly the hepatitis B surface and envelope antigen levels in HepG2.2.15 cells and produced the maximum inhibition rates of 79.94% and 76.41%, respectively. Meanwhile, test compound at 50-100 µg/mL inhibited markedly HBV DNA replication. In conclusion, this study verifies the anti-hepatitis B effect of cichoric acid from Cichorium intybus leaves. In addition, cichoric acid could be used to design the antiviral agents.


Assuntos
Ácidos Cafeicos/farmacocinética , Chicória/química , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Succinatos/farmacocinética , Animais , Ácidos Cafeicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , DNA Viral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Patos , Galactosamina/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B do Pato/genética , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Hepatócitos/virologia , Humanos , Estrutura Molecular , Folhas de Planta/química , Substâncias Protetoras/isolamento & purificação , Succinatos/isolamento & purificação , Replicação Viral/efeitos dos fármacos
13.
World J Gastroenterol ; 20(2): 436-44, 2014 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-24574712

RESUMO

The hepatitis B virus (HBV) is considered to be a major public health problem worldwide, and a significant number of reports on nosocomial outbreaks of HBV infections have been reported. Prevention of indirect HBV transmission by contaminated objects is only possible through the use of infection-control principles, including the use of chemical biocides, which are proven to render the virus non-infectious. The virucidal activity of biocides against HBV cannot be predicted; therefore, validation of the virucidal action of disinfectants against HBV is essential. However, feasible HBV infectivity assays have not yet been established. Thus, surrogate models have been proposed for testing the efficacy of biocides against HBV. Most of these assays do not correlate with HBV infectivity. Currently, the most promising and feasible assay is the use of the taxonomically related duck hepatitis B virus (DHBV), which belongs to the same Hepadnaviridae virus family. This paper reviews the application of DHBV, which can be propagated in vitro in primary duck embryonic hepatocytes, for the testing of biocides and describes why this model can be used as reliable method to evaluate disinfectants for efficacy against HBV. The susceptibility levels of important biocides, which are often used as ingredients for commercially available disinfectants, are also described.


Assuntos
Desinfetantes/farmacologia , Desinfecção/métodos , Contaminação de Equipamentos/prevenção & controle , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/prevenção & controle , Animais , Células Cultivadas , Hepatite B/transmissão , Hepatite B/virologia , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Vírus da Hepatite B do Pato/patogenicidade , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/patogenicidade , Humanos , Reprodutibilidade dos Testes , Cultura de Vírus
14.
Mol Med Rep ; 9(4): 1381-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24481875

RESUMO

In order to validate the antiviral effect against hepatitis B virus (HBV) of Taraxacum mongolicum (T. mongolicum), the protective effect on hepatocytes, and antiviral properties against duck hepatitis B virus (DHBV) and HBV of T. mongolicum extract (TME) were evaluated in chemically-injured neonatal rat hepatocytes, DHBV-infected duck fetal hepatocytes and HBV-transfected HepG2.2.15 cells, respectively. The results demonstrated that TME at 50-100 µg/ml improved D-galactosamine (D-GalN), thioacetamide (TAA) and tert-butyl hydroperoxide (t-BHP)-injured rat hepatocytes, and produced protection rates of 42.2, 34.6 and 43.8% at 100 µg/ml, respectively. Furthermore, TME at 1-100 µg/ml markedly inhibited DHBV DNA replication. Additionally, TME at 25-100 µg/ml reduced HBsAg and HBeAg levels and produced inhibition rates of 91.39 and 91.72% at 100 µg/ml, respectively. TME markedly inhibited HBV DNA replication at 25-100 µg/ml. The results demonstrate the potent antiviral effect of T. mongolicum against HBV effect. The protective of TME effect on hepatocytes may be achieved by its ability to ameliorate oxidative stress. The antiviral properties of TME may contribute to blocking protein synthesis steps and DNA replication. Furthermore, major components of TME were quantificationally analyzed. These data provide scientific evidence supporting the traditional use of TME in the treatment of hepatitis.


Assuntos
Antivirais/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Animais , Animais Recém-Nascidos , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Patos , Galactosamina , Glucosídeos/química , Glucosídeos/farmacologia , Células Hep G2 , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B do Pato/fisiologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Luteolina/química , Luteolina/farmacologia , Ratos , Ratos Sprague-Dawley , Tioacetamida , terc-Butil Hidroperóxido
15.
Acta Pharmacol Sin ; 35(3): 410-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24487969

RESUMO

AIM: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. METHODS: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg⁻¹·d⁻¹) for 15 d. RESULTS: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 µmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 µmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 µmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. CONCLUSION: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.


Assuntos
Antivirais/farmacologia , Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , RNA Viral/efeitos dos fármacos , Tiazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Farmacorresistência Viral Múltipla/genética , Patos , Células Hep G2 , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/genética , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Vírus da Hepatite B/genética , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite Viral Animal/virologia , Humanos , Mutação , Nucleocapsídeo/metabolismo , RNA Viral/biossíntese , Fatores de Tempo , Transfecção
16.
Bull Exp Biol Med ; 155(6): 821-4, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24288775

RESUMO

The virucidal action of solvent tributyl phosphate and detergent sodium cholate used in the production of immunoglobulin for inactivation of viruses with lipid envelope was studied on the model of duck hepatitis B virus. PCR analysis revealed no significant decrease in duck hepatitis B virus DNA concentrations after treatment with solvent/detergent. At the same time, in vivo experiments showed that treatment of duck hepatitis B virus with tributyl phosphate (concentration >0.15%) and sodium cholate (concentration >0.1%) at 37°C for 6 h or longer completely inactivated this model virus added to immunoglobulin solution in concentration 5 log ID50. Duck hepatitis B virus appears to be one of the most acceptable model viruses for validation of virus inactivating technologies in manufacturing human plasma preparations.


Assuntos
Desinfetantes/farmacologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Imunoglobulina G/isolamento & purificação , Organofosfatos/farmacologia , Colato de Sódio/farmacologia , Inativação de Vírus , Animais , Patos , Humanos , Soros Imunes/isolamento & purificação , Soluções
17.
Virology ; 446(1-2): 357-64, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24074600

RESUMO

Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis.


Assuntos
DNA Viral/isolamento & purificação , Guanina/análogos & derivados , Vírus da Hepatite B do Pato/fisiologia , Hepatócitos/virologia , Fígado/virologia , Mitose , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , DNA Circular/isolamento & purificação , Patos , Guanina/administração & dosagem , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatócitos/fisiologia
18.
PLoS Pathog ; 9(9): e1003613, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24068929

RESUMO

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of infected hepatocyte and serves as the transcriptional template for viral mRNA synthesis. Elimination of cccDNA is the prerequisite for either a therapeutic cure or immunological resolution of HBV infection. Although accumulating evidence suggests that inflammatory cytokines-mediated cure of virally infected hepatocytes does occur and plays an essential role in the resolution of an acute HBV infection, the molecular mechanism by which the cytokines eliminate cccDNA and/or suppress its transcription remains elusive. This is largely due to the lack of convenient cell culture systems supporting efficient HBV infection and cccDNA formation to allow detailed molecular analyses. In this study, we took the advantage of a chicken hepatoma cell line that supports tetracycline-inducible duck hepatitis B virus (DHBV) replication and established an experimental condition mimicking the virally infected hepatocytes in which DHBV pregenomic (pg) RNA transcription and DNA replication are solely dependent on cccDNA. This cell culture system allowed us to demonstrate that cccDNA transcription required histone deacetylase activity and IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of acetylated histone H3 lysine 9 (H3K9) and 27 (H3K27) in cccDNA minichromosomes. Moreover, IFN-α treatment also induced a delayed response that appeared to accelerate the decay of cccDNA. Our studies have thus shed light on the molecular mechanism by which IFN-α noncytolytically controls hepadnavirus infection.


Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Epigênese Genética , Vírus da Hepatite B do Pato/metabolismo , Hepatócitos/virologia , Interferon-alfa/metabolismo , Transcrição Genética , Acetilação/efeitos dos fármacos , Animais , Proteínas Aviárias/antagonistas & inibidores , Proteínas Aviárias/biossíntese , Proteínas Aviárias/metabolismo , Linhagem Celular , Galinhas , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Infecções por Hepadnaviridae/metabolismo , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Histonas/metabolismo , Lisina/química , Biossíntese de Proteínas/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Transcrição Genética/efeitos dos fármacos
19.
Antimicrob Agents Chemother ; 57(11): 5291-8, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23939902

RESUMO

Nucleic acid polymers (NAPs) utilize the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) to target protein interactions involved in viral replication. NAPs are broadly active against a diverse range of enveloped viruses that use type I entry mechanisms. The antiviral activity of NAPs against hepatitis B virus (HBV) infection was assessed in vitro in duck hepatitis B virus (DHBV)-infected primary duck hepatocytes (PDH). NAPs efficiently entered PDH in the absence of any transfection agent and displayed antiviral activity at concentrations of 0.01 to 10 µM, measured by their ability to prevent the intracellular accumulation of DHBV surface antigen, which was independent of their nucleotide sequence and was specifically dependent on phosphorothioation. Higher levels of antiviral activity were observed with NAPs 40 nucleotides in length or longer. The fully degenerate NAP (REP 2006) was active during DHBV infection or when added 12 h after infection. In contrast, an acidic-pH-sensitive NAP (REP 2031) that was broadly active against other viruses displayed antiviral activity when present during DHBV infection but no activity when added 12 h after infection, suggesting that NAPs exert their postentry effect in an acidic environment unique to DHBV infection. Both REP 2006 and REP 2031 displayed negligible cytotoxicity in PDH at concentrations of up to 10 µM, as assessed using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cytotoxicity assay. The antiviral activity of NAPs against DHBV in vitro was strictly dependent on their amphipathic character, suggesting that NAPs interact with amphipathic target(s) that are important for DHBV entry and postentry mechanisms required for infection.


Assuntos
Antivirais/farmacologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Oligonucleotídeos Fosforotioatos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/síntese química , Sobrevivência Celular/efeitos dos fármacos , Antígenos de Superfície da Hepatite B/biossíntese , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Hepatócitos/virologia , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos Fosforotioatos/síntese química , Cultura Primária de Células , Relação Estrutura-Atividade
20.
Antimicrob Agents Chemother ; 57(11): 5299-306, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23939904

RESUMO

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks. The degenerate NAP REP 2006 prevented the development of widespread and persistent DHBV infection in 14-day-old ducks, while the acidic-pH-sensitive NAP REP 2031 had little or no prophylactic effect. REP 2006 displayed significant toxicity in ducks, which was attributed to CpG-mediated proinflammation, while REP 2031 (which has no CpG motifs) displayed no toxicity. A third NAP, REP 2055, which was designed to retain amphipathic activity at acidic pH and contained no CpG motifs, was well tolerated and displayed prophylactic activity against DHBV infection at doses as low as 1 mg/kg of body weight/day. These studies suggest that NAPs can be easily and predictably tailored to retain anti-DHBV activity and to have minimal toxic effects in vivo. Future studies are planned to establish the therapeutic efficacy of NAPs against persistent DHBV infection.


Assuntos
Antivirais/farmacologia , Infecções por Hepadnaviridae/tratamento farmacológico , Vírus da Hepatite B do Pato/efeitos dos fármacos , Hepatite Viral Animal/tratamento farmacológico , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Antivirais/síntese química , Ilhas de CpG , Esquema de Medicação , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/crescimento & desenvolvimento , Hepatite Viral Animal/virologia , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos Fosforotioatos/síntese química , Relação Estrutura-Atividade , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
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