AIM To assess daclatasvir plus asunaprevir(d UAL) in treatment-na?ve patients from China's Mainland, Russia and South Korea with hepatitis C virus(HCV) genotype 1 b infection. METHODS Patients were randomly assign...AIM To assess daclatasvir plus asunaprevir(d UAL) in treatment-na?ve patients from China's Mainland, Russia and South Korea with hepatitis C virus(HCV) genotype 1 b infection. METHODS Patients were randomly assigned(3:1) to receive 24 wk of treatment with d UAL(daclatasvir 60 mg once daily and asunaprevir 100 mg twice daily) beginning on day 1 of the treatment period(immediate treatment arm) or following 12 wk of matching placebo(placebodeferred treatment arm). The primary endpoint was a comparison of sustained virologic response at posttreatment week 12(SVR12) compared with the historical SVR rate for peg-interferon plus ribavirin(70%) among patients in the immediate treatment arm. The first 12 wk of the study were blinded. Safety was assessed in d UAL-treated patients compared with placebo patients during the first 12 wk(doubleblind phase), and during 24 wk of d UAL in both arms combined.RESULTS In total, 207 patients were randomly assigned to immediate(n = 155) or placebo-deferred(n = 52) treatment. Most patients were Asian(86%), female(59%) and aged < 65 years(90%). Among them, 13% had cirrhosis, 32% had IL28 B non-CC genotypes and 53% had baseline HCV RNA levels of ≥ 6 million IU/m L. Among patients in the immediate treatment arm, SVR12 was achieved by 92%(95% confidence interval: 87.2-96.0), which was significantly higher than the historical comparator rate(70%). SVR12 was largely unaffected by cirrhosis(89%), age ≥ 65 years(92%), male sex(90%), baseline HCV RNA ≥ 6 million(89%) or IL28 B non-CC genotypes(96%), although SVR12 was higher among patients without(96%) than among those with(53%) baseline NS5 A resistanceassociated polymorphisms(at L31 or Y93 H). during the double-blind phase, aminotransferase elevations were more common among placebo recipients than among patients receiving d UAL. during 24 wk of d UAL therapy(combined arms), the most common adverse events(≥ 10%) were elevated alanine aminotransferase and upper respiratory tract infection; emergent grade 3-4 laboratory abnormalities were展开更多
AIM: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Althou...AIM: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA- dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear. METHODS: HCV quasispecies were studied by clon- ing and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-α2b for 3 mo followed by IFN-α2b alone or com- bined IFN-R therapy for 9 additional months. Patients were categorized intro two groups based on their re- sponse to the treatments: 7 with sustained virological re- sponse (SVR) (quasispecies = 150) and 3 non-respond- ers (NR) to IFN-R (quasispecies = 106). RESULTS: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispe- cies during therapy. Analysis of amino acids substitu- tions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate thetwo groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients. CONCLUSION: These results suggest that detailed mo- lecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.展开更多
The innate immune response is triggered by a variety of pathogens, including viruses, and requires rapid induction of typeⅠ?interferons (IFN), such as IFNβ and IFNα. IFN induction occurs when specific pathogen moti...The innate immune response is triggered by a variety of pathogens, including viruses, and requires rapid induction of typeⅠ?interferons (IFN), such as IFNβ and IFNα. IFN induction occurs when specific pathogen motifs bind to specific cellular receptors. In non-professional immune, virally-infected cells, IFN induction is essentially initiated after the binding of dsRNA structures to TLR3 receptors or to intracytosolic RNA helicases, such as RIG-Ⅰ/MDA5. This leads to the recruitment of specific adaptors, such as TRIF for TLR3 and the mitochondrial-associated IPS-1/VISA/MAVS/CARDIF adapter protein for the RNA helicases, and the ultimate recruitment of kinases, such as MAPKs, the canonical IKK complex and the TBK1/IKKε kinases, which activate the transcription factors ATF-2/ c-jun, NF-κB and IRF3, respectively. The coordinated action of these transcription factors leads to induction of IFN and of pro-inflammatory cytokines and to the establishment of the innate immune response. HCV can cleave both the adapters TRIF and IPS-1/VISA/MAVS/ CARDIF through the action of its NS3/4A protease. This provokes abrogation of the induction of the IFN and cytokine pathways and favours viral propagation and presumably HCV chronic infection.展开更多
AIM:To study the effect of hepatitis C virus nonstructural region 3 (HCV NS3) protein on proliferation and transformation of normal human liver cell line. METHODS: QSG7701 cells were transfected with pRcHCNS3-5', ...AIM:To study the effect of hepatitis C virus nonstructural region 3 (HCV NS3) protein on proliferation and transformation of normal human liver cell line. METHODS: QSG7701 cells were transfected with pRcHCNS3-5', pRcHCNS3-3'and pRcCMV using lipofectamine transfecting technique and selected with G418 method. Expression of HCV NS3 protein was determined by immunohistochemistry. Biologic characteristics of transfected cells were evaluated by population doubling time and soft agar assays. Activation of MAPK was analyzed using Western blot with phosphospecific monoclonal antibody against dually phosphorylated MAPK. RESULTS: QSG7701 cells transfected with pRcHCNS3-5' showed strong intracellular expression of HCVNS3 protein, and the positive signal was localized in cytoplasm.The expressing strength of HCVNS3 protein in pRcHCNS3-3'-transfected cells was weaker than that in pRcHCNS3-5'-transfected cells. The population doubling time in the transfected cells with pRcHCNS3-5' (12 h) was much shorter than those with pRcHCNS3-3', pRcCMV and normal cells (24, 26, 28 h, respectively) (P<0.01). The transfected cells with pRcHCNS3-5' showed much more anchorage independent colonies than that in those with pRcHCNS3-3' and pRcCMV (P<0.01). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcHCNS3-3', pRcCMV and controls were 33%, 1.33%, 1.46%, 1.11% respectively. The level of phosphorylated MAPK in the cells with pRcHCNS3-5' was much higher than that in those with pRcHCNS3-3'and pRcCMV and normal cells (P<0.01). CONCLUSION: The results suggest that (1) QSG7701 cells are a better human liver cell line for investigating' the pathogenesis of HCV NS3 protein. (2) 5' region of the HCV genome segment encoding HCV NS3 is involved in cell growth and cell phenotype. (3) HCV NS3 N-terminal peptide may up-regulate the activation of MAPK, but not affect the expression of MAPK.展开更多
The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The...The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.展开更多
The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects...The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.展开更多
AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence...AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.展开更多
文摘AIM To assess daclatasvir plus asunaprevir(d UAL) in treatment-na?ve patients from China's Mainland, Russia and South Korea with hepatitis C virus(HCV) genotype 1 b infection. METHODS Patients were randomly assigned(3:1) to receive 24 wk of treatment with d UAL(daclatasvir 60 mg once daily and asunaprevir 100 mg twice daily) beginning on day 1 of the treatment period(immediate treatment arm) or following 12 wk of matching placebo(placebodeferred treatment arm). The primary endpoint was a comparison of sustained virologic response at posttreatment week 12(SVR12) compared with the historical SVR rate for peg-interferon plus ribavirin(70%) among patients in the immediate treatment arm. The first 12 wk of the study were blinded. Safety was assessed in d UAL-treated patients compared with placebo patients during the first 12 wk(doubleblind phase), and during 24 wk of d UAL in both arms combined.RESULTS In total, 207 patients were randomly assigned to immediate(n = 155) or placebo-deferred(n = 52) treatment. Most patients were Asian(86%), female(59%) and aged < 65 years(90%). Among them, 13% had cirrhosis, 32% had IL28 B non-CC genotypes and 53% had baseline HCV RNA levels of ≥ 6 million IU/m L. Among patients in the immediate treatment arm, SVR12 was achieved by 92%(95% confidence interval: 87.2-96.0), which was significantly higher than the historical comparator rate(70%). SVR12 was largely unaffected by cirrhosis(89%), age ≥ 65 years(92%), male sex(90%), baseline HCV RNA ≥ 6 million(89%) or IL28 B non-CC genotypes(96%), although SVR12 was higher among patients without(96%) than among those with(53%) baseline NS5 A resistanceassociated polymorphisms(at L31 or Y93 H). during the double-blind phase, aminotransferase elevations were more common among placebo recipients than among patients receiving d UAL. during 24 wk of d UAL therapy(combined arms), the most common adverse events(≥ 10%) were elevated alanine aminotransferase and upper respiratory tract infection; emergent grade 3-4 laboratory abnormalities were
基金Supported by a grant from l’Agence National de la Recherche sur le Sida (ANRS grant 2001/011)
文摘AIM: To evaluate the implication of substitutions in the hepatitis C virus (HCV) non-structural 5A (NS5A) protein in the resistance of HCV during mono-interferon (IFN) or combined IFN-ribavirin (IFN-R) therapy. Although NS5A has been reported to interact with the HCV RNA- dependent RNA polymerase, NS5B, as well as with many cellular proteins, the function of NS5A in the life cycle of HCV remains unclear. METHODS: HCV quasispecies were studied by clon- ing and sequencing of sequential isolates from patients infected by HCV genotype 1b. Patients were treated by IFN-α2b for 3 mo followed by IFN-α2b alone or com- bined IFN-R therapy for 9 additional months. Patients were categorized intro two groups based on their re- sponse to the treatments: 7 with sustained virological re- sponse (SVR) (quasispecies = 150) and 3 non-respond- ers (NR) to IFN-R (quasispecies = 106). RESULTS: Prior to treatment, SVR patients displayed a lower complexity of quasispecies than NR patients. Most patients had a decrease in the complexity of quasispe- cies during therapy. Analysis of amino acids substitu- tions showed that the degree of the complexity of the interferon sensitivity-determining region (ISDR) and the V3 domain of NS5A protein was able to discriminate thetwo groups of patients. Moreover, SVR patients displayed more variability in the NS5A region than NR patients. CONCLUSION: These results suggest that detailed mo- lecular analysis of the NS5A region may be important for understanding its function in IFN response during HCV 1b infection.
基金grants from the Agence Nationale pour la Recherche contre le SIDA
文摘The innate immune response is triggered by a variety of pathogens, including viruses, and requires rapid induction of typeⅠ?interferons (IFN), such as IFNβ and IFNα. IFN induction occurs when specific pathogen motifs bind to specific cellular receptors. In non-professional immune, virally-infected cells, IFN induction is essentially initiated after the binding of dsRNA structures to TLR3 receptors or to intracytosolic RNA helicases, such as RIG-Ⅰ/MDA5. This leads to the recruitment of specific adaptors, such as TRIF for TLR3 and the mitochondrial-associated IPS-1/VISA/MAVS/CARDIF adapter protein for the RNA helicases, and the ultimate recruitment of kinases, such as MAPKs, the canonical IKK complex and the TBK1/IKKε kinases, which activate the transcription factors ATF-2/ c-jun, NF-κB and IRF3, respectively. The coordinated action of these transcription factors leads to induction of IFN and of pro-inflammatory cytokines and to the establishment of the innate immune response. HCV can cleave both the adapters TRIF and IPS-1/VISA/MAVS/ CARDIF through the action of its NS3/4A protease. This provokes abrogation of the induction of the IFN and cytokine pathways and favours viral propagation and presumably HCV chronic infection.
基金Supported by the National Natural Science Foundation of China,No. 30270601
文摘AIM:To study the effect of hepatitis C virus nonstructural region 3 (HCV NS3) protein on proliferation and transformation of normal human liver cell line. METHODS: QSG7701 cells were transfected with pRcHCNS3-5', pRcHCNS3-3'and pRcCMV using lipofectamine transfecting technique and selected with G418 method. Expression of HCV NS3 protein was determined by immunohistochemistry. Biologic characteristics of transfected cells were evaluated by population doubling time and soft agar assays. Activation of MAPK was analyzed using Western blot with phosphospecific monoclonal antibody against dually phosphorylated MAPK. RESULTS: QSG7701 cells transfected with pRcHCNS3-5' showed strong intracellular expression of HCVNS3 protein, and the positive signal was localized in cytoplasm.The expressing strength of HCVNS3 protein in pRcHCNS3-3'-transfected cells was weaker than that in pRcHCNS3-5'-transfected cells. The population doubling time in the transfected cells with pRcHCNS3-5' (12 h) was much shorter than those with pRcHCNS3-3', pRcCMV and normal cells (24, 26, 28 h, respectively) (P<0.01). The transfected cells with pRcHCNS3-5' showed much more anchorage independent colonies than that in those with pRcHCNS3-3' and pRcCMV (P<0.01). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcHCNS3-3', pRcCMV and controls were 33%, 1.33%, 1.46%, 1.11% respectively. The level of phosphorylated MAPK in the cells with pRcHCNS3-5' was much higher than that in those with pRcHCNS3-3'and pRcCMV and normal cells (P<0.01). CONCLUSION: The results suggest that (1) QSG7701 cells are a better human liver cell line for investigating' the pathogenesis of HCV NS3 protein. (2) 5' region of the HCV genome segment encoding HCV NS3 is involved in cell growth and cell phenotype. (3) HCV NS3 N-terminal peptide may up-regulate the activation of MAPK, but not affect the expression of MAPK.
文摘The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.
文摘The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.
文摘AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.
