A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infecte...A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.展开更多
AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patie...AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.展开更多
AIM: To evaluate the antiviral potency of a new antihepatitis C virus(HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in...AIM: To evaluate the antiviral potency of a new antihepatitis C virus(HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices(2.7 × 106 cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant(multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription- polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS: The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dosedependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect(compared to hydroxychloroquine EC50 = 1.17 μmol/L).CONCLUSION: Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dosedependent manner without cytotoxic effect.展开更多
根据已发表的鸭甲肝病毒(Duck hepatitis A virus,DHAV)疫苗株A66株基因组序列,利用生物学分析软件DNAStar进行VP1蛋白的二级结构和抗原域预测,确定其主要抗原域(major antigen domain of VP1,VP1M),设计并合成一对可扩增VP1M的特异性引...根据已发表的鸭甲肝病毒(Duck hepatitis A virus,DHAV)疫苗株A66株基因组序列,利用生物学分析软件DNAStar进行VP1蛋白的二级结构和抗原域预测,确定其主要抗原域(major antigen domain of VP1,VP1M),设计并合成一对可扩增VP1M的特异性引物,PCR扩增获得长为327 bp的VP1M基因片段。将VP1M基因片段定向克隆至p GEX-4T-1表达载体中,鉴定正确后进行IPTG诱导表达,获得了以包涵体为主的重组蛋白。重组蛋白纯化后,经过Western blot检测表明重组蛋白具有良好的抗原性。以该蛋白作为包被抗原,成功建立了检测DHAV-1的间接ELISA方法。该方法具有良好的特异性、敏感性和重复性,为DHAV的快速诊断、流行病学调查和免疫鸭群的抗体检测提供了快速实用的检测手段。展开更多
Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specifici...Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis. Methods Samples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study. Results Only 48 （1.3%） of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 （24.3%） of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 （60%） were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% （20/23） and 69.6% （16/23） respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test （r=-0.989, P〈0.05）, and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV Utres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually. Conclusions The HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.展开更多
文摘A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.
基金Suppprted by the Mational Natural Science Foundation of China,No.39670672.
文摘AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.
基金Supported by The Institut National de la Sante et de la Recherche Medicale(INSERM,France)the personal support of Professor Jean-Francois Delfraissy as Director of the French Agency,Agence Nationale de Recherches sur le Sida et les hepatites virales(ANRS)
文摘AIM: To evaluate the antiviral potency of a new antihepatitis C virus(HCV) antiviral agent targeting the cellular autophagy machinery. METHODS: Non-infected liver slices, obtained from human liver resection and cut in 350 μm-thick slices(2.7 × 106 cells per slice) were infected with cell culture-grown HCV Con1b/C3 supernatant(multiplicity of infection = 0.1) cultivated for up to ten days. HCV infected slices were treated at day 4 post-infection with GNS-396 for 6 d at different concentrations. HCV replication was evaluated by strand-specific real-time quantitative reverse transcription- polymerase chain reaction. The infectivity titers of supernatants were evaluated by foci formation upon inoculation into naive Huh-7.5.1 cells. The cytotoxic effect of the drugs was evaluated by lactate dehydrogenase leakage assays. RESULTS: The antiviral efficacy of a new antiviral drug, GNS-396, an autophagy inhibitor, on HCV infection of adult human liver slices was evidenced in a dosedependent manner. At day 6 post-treatment, GNS-396 EC50 was 158 nmol/L without cytotoxic effect(compared to hydroxychloroquine EC50 = 1.17 μmol/L).CONCLUSION: Our results demonstrated that our ex vivo model is efficient for evaluation the potency of autophagy inhibitors, in particular a new quinoline derivative GNS-396 as antiviral could inhibit HCV infection in a dosedependent manner without cytotoxic effect.
基金the Capital Medical Developing Scientific and Research Fund (No. 2002-3068)the Basic and Clinical Joint Project of Capital Medical University (No.02JL20)
文摘Background Hepatitis C virus （HCV） core antigen assays have been produced to exclude infectious donations collected during the preseroconversion window phase （PWP）. For the same purpose, we evaluated the specificity and sensitivity of a novel hepatitis C virus NS3 antigen detection immunoassay and the application of this assay in clinical diagnosis. Methods Samples from 77 healthy subjects, 173 anti-HCV positive patients and 3708 hepatitis patients other than HCV positive were tested with the HCV NS3 antigen assay. Some HCV NS3 antigen positive samples were further validated with HCV-RNA, neutralization and immunodot assays. Twenty-five sequential samples from 11 HCV NS3 antigen positive patients were subjected to kinetic study. Results Only 48 （1.3%） of 3708 anti-HCV negative samples were positive for HCV NS3 antigen. Among them, 44 of 3030 samples from patients only infected with HBV were HCV NS3 antigen positive, 4 of the 445 samples from patients infected with other type hepatitis were HCV NS3 antigen positive. In addition, 42 （24.3%） of 173 anti-HCV positive samples were HCV NS3 antigen positive and all 77 samples from healthy subjects were negative to HCV NS3 antigen assay. Of the 15 HCV NS3 antigen positive samples, 9 （60%） were HCV-RNA positive. The neutralization and positive percentage of immunodot assay for 23 HCV NS3 antigen positive sera were 87.0% （20/23） and 69.6% （16/23） respectively. Of the 25 sequential samples from 11 HCV NS3 antigen positive patients, there was a negative correlation between the OD values and the duration of test （r=-0.989, P〈0.05）, and there were correlations among their HCV NS3 antigen, HCV-RNA and anti-HCV Utres. The anti-HCV antibodies of two sera were detected while their OD values of HCV NS3 antigen decreased gradually. Conclusions The HCV NS3 antigen detection assay showed perfect specificity and high sensitivity. Thus, it would be useful and economical as a routine test in laboratories for early diagnosis of HCV infection and prevention.
