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1.
Braz J Infect Dis ; 22(6): 477-486, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30586543

RESUMO

Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-ß-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.


Assuntos
Antivirais/farmacologia , Citidina/análogos & derivados , Farmacorresistência Viral/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Lamivudina/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , DNA Viral/química , Feminino , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação
2.
Braz. j. infect. dis ; Braz. j. infect. dis;22(6): 477-486, Nov.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-984016

RESUMO

ABSTRACT Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.


Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Antivirais/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Lamivudina/farmacologia , Citidina/análogos & derivados , DNA Viral/química , Testes de Sensibilidade Microbiana , Linhagem Celular , Vírus da Hepatite B/isolamento & purificação , Vírus da Hepatite B/fisiologia , Hepatócitos/virologia , Farmacorresistência Viral/efeitos dos fármacos , Mutação
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