Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus classified as a biosafety level-4(BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccine...Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus classified as a biosafety level-4(BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion(OHFVDNS1) and reporter virus replacing NS1 with the Gaussia luciferase(Gluc)(OHFV-ΔNS1-Gluc). Both the defective OHFVDNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1(BHK21NS1) but not in na?ve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z0 value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.展开更多
Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibi...Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors.In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA,which expresses Gaussia luciferase upon influenza A virus infection.Using this cell line,an assay was developed and optimized to search for inhibitors of influenza virus replication.Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format(Z 0 factor value40.8).A pilot screening provides further evidence for validation of the assay.Taken together,this work provides a simple,convenient,and reliable HTS assay to identify compounds with anti-influenza activity.展开更多
Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology.However,in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably ref...Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology.However,in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs.Sensors for miRNA were developed by adding miRNA target sequences to the 3'-untranslated region of Gaussia princeps luciferase (Gluc) mRNA.These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood.Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells,but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing.Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities.We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122,miR142 and miR-34a activities,as well as for exogenous miR-34a activity.Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.展开更多
Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigen...Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.展开更多
Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia lucifer...Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.展开更多
活体动物microRNA(miRNA)检测技术对阐明miRNA的生物学功能非常重要.但是,目前缺乏一种方便灵敏的实时反映活体动物miRNA活性及其变化的体外监测手段.通过在分泌型荧光素酶Gluc(Gaussia princeps luciferase)mRNA的3′非翻译区插入相应m...活体动物microRNA(miRNA)检测技术对阐明miRNA的生物学功能非常重要.但是,目前缺乏一种方便灵敏的实时反映活体动物miRNA活性及其变化的体外监测手段.通过在分泌型荧光素酶Gluc(Gaussia princeps luciferase)mRNA的3′非翻译区插入相应miRNA的靶序列构建一系列miRNA传感器.将miRNA传感器体外转染培养细胞和通过水动力注射方法转染小鼠肝脏,通过检测细胞培养上清和外周血液中的Gluc来反映细胞内和活体动物肝脏的miRNA活性.结果显示,CMV启动子驱动的miRNA传感器可以长期监测体外培养细胞中的miRNA和短期监测小鼠肝脏的miRNA,但由于启动子在肝脏的沉默不能用于长期监测肝脏miRNA;CAG启动子驱动的传感器则成功地实现了对肝脏内源性miR-122,miR-142和miR-34a以及对肝脏过表达的miR-34a的长期实时监测.利用以Gluc为报告基因的miRNA传感器,通过检测外周血Gluc可以方便地在体外长期连续监测小鼠肝脏的miRNA.展开更多
利用混合高斯模型(gaussian mixture model,GMM)学习自然图像块的纹理结构,提出一种基于图像块先验的低秩近似和维纳滤波的去噪算法。该算法能够同时利用外部图像块的先验结构信息和内部图像的自相似性,对待去噪图像进行分块聚类,并根...利用混合高斯模型(gaussian mixture model,GMM)学习自然图像块的纹理结构,提出一种基于图像块先验的低秩近似和维纳滤波的去噪算法。该算法能够同时利用外部图像块的先验结构信息和内部图像的自相似性,对待去噪图像进行分块聚类,并根据每类相似块的数量进行协同滤波。当相似图像块数量较多时,采用低秩近似的方法复原,有效利用图像的内部自相似性;当相似图像块数量较少时,采用维纳滤波,利用先验信息保持图像重要的纹理结构。试验结果表明此方法较适用于弧形边界和角点等存在较少相似块的自然图像,其峰值信噪比(peak signal to noise ratio,PSNR)和视觉效果优于目前部分主流算法。展开更多
基金supported by National Science and Technology Major Project on Important Infectious Diseases Prevention and Control (2018ZX10734404-010)National Key Research and Development Program of China (2018YFA0507201)
文摘Omsk hemorrhagic fever virus(OHFV) is a tick-borne flavivirus classified as a biosafety level-4(BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion(OHFVDNS1) and reporter virus replacing NS1 with the Gaussia luciferase(Gluc)(OHFV-ΔNS1-Gluc). Both the defective OHFVDNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1(BHK21NS1) but not in na?ve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z0 value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.
基金This work was supported in part by National S&T Major Special Project on Major New Drug Innovation(No.2012ZX09301-002-004)(S.C.)National Science and Technology Major Project,“China Mega-Project for Infectious Disease”(No.2013ZX 10004601-002),and the Xiehe Scholar(S.C.).
文摘Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus.Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors.In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA,which expresses Gaussia luciferase upon influenza A virus infection.Using this cell line,an assay was developed and optimized to search for inhibitors of influenza virus replication.Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format(Z 0 factor value40.8).A pilot screening provides further evidence for validation of the assay.Taken together,this work provides a simple,convenient,and reliable HTS assay to identify compounds with anti-influenza activity.
基金supported by the China Special Key Program on Infectious Diseases (Grant Nos.2008ZX10002-023 and 2008ZX10001-012)the Research Program from the State Key Laboratory of Molecular Virology and Genetic Engineering (Grant No.2008-S-0003)
文摘Technology for monitoring in vivo microRNA (miRNA) activity is extremely important for elucidating miRNA biology.However,in vivo studies of miRNA have been hampered by the lack of a convenient approach to reliably reflect real-time functional changes in miRNAs.Sensors for miRNA were developed by adding miRNA target sequences to the 3'-untranslated region of Gaussia princeps luciferase (Gluc) mRNA.These sensors were then evaluated in vitro and in vivo by measuring Gluc activity in cell supernatants and in peripheral blood.Sensors driven by the CMV promoter were effective for monitoring miR-122 in living cells,but not for the long-term monitoring of miR-122 or miR-142 in mouse liver because of CMV-promoter silencing.Replacing the CMV promoter with a CAG promoter rendered these sensors effective for the long-term monitoring of relevant liver miRNA activities.We subsequently used the CAG-promoter-based sensor for the long-term monitoring of endogenous liver miR-122,miR142 and miR-34a activities,as well as for exogenous miR-34a activity.Our study demonstrates that real-time in vivo activities of miRNAs can be continuously and conveniently detected in mouse liver using the sensors that we have developed.
基金supported by National Major Scientific and Technological Special Project for ‘‘AIDS and Viral Hepatitis and Other Major Infectious Diseases Prevention and Control’’ during the Twelfth Five-year Plan Period (No. 2013ZX10004-601)
文摘Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.
基金Supported by National High Technology Research and Development Program of China (863 Program) (2007AA021206,2007AA021106)
文摘Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.
文摘活体动物microRNA(miRNA)检测技术对阐明miRNA的生物学功能非常重要.但是,目前缺乏一种方便灵敏的实时反映活体动物miRNA活性及其变化的体外监测手段.通过在分泌型荧光素酶Gluc(Gaussia princeps luciferase)mRNA的3′非翻译区插入相应miRNA的靶序列构建一系列miRNA传感器.将miRNA传感器体外转染培养细胞和通过水动力注射方法转染小鼠肝脏,通过检测细胞培养上清和外周血液中的Gluc来反映细胞内和活体动物肝脏的miRNA活性.结果显示,CMV启动子驱动的miRNA传感器可以长期监测体外培养细胞中的miRNA和短期监测小鼠肝脏的miRNA,但由于启动子在肝脏的沉默不能用于长期监测肝脏miRNA;CAG启动子驱动的传感器则成功地实现了对肝脏内源性miR-122,miR-142和miR-34a以及对肝脏过表达的miR-34a的长期实时监测.利用以Gluc为报告基因的miRNA传感器,通过检测外周血Gluc可以方便地在体外长期连续监测小鼠肝脏的miRNA.
文摘利用混合高斯模型(gaussian mixture model,GMM)学习自然图像块的纹理结构,提出一种基于图像块先验的低秩近似和维纳滤波的去噪算法。该算法能够同时利用外部图像块的先验结构信息和内部图像的自相似性,对待去噪图像进行分块聚类,并根据每类相似块的数量进行协同滤波。当相似图像块数量较多时,采用低秩近似的方法复原,有效利用图像的内部自相似性;当相似图像块数量较少时,采用维纳滤波,利用先验信息保持图像重要的纹理结构。试验结果表明此方法较适用于弧形边界和角点等存在较少相似块的自然图像,其峰值信噪比(peak signal to noise ratio,PSNR)和视觉效果优于目前部分主流算法。
