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Hepatitis C virus NS3/4A with sequence variation at amino-terminus has different serine protease activities and inhibitory activities on IFN-β induction and p53-dependent transcriptional activation

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Journal of Nanjing Medical University 2009年第4期23卷 257-264页

作者： Xueping Wang Fujun Li Motoko Nagano-Fujii Lin Deng Kikumi Kitayama hak hotta Editorial Department of Journal of Fourth Military Medical University Xi' an 710033 China Department of Medical Affairs Administration Lanzhou General Hospital Lanzhou Military Command Lanzhou 730050 China Division of Microbiology Kobe University Graduate School of Medicine Kobe 650-0017 Japan

Objective： To construct the point mutation plasmids expressing HCV NS3/4A with different secondary structures at the N-terminus, and to analyze their serine protease activities. Methods： The point mutation plasmid constructs were generated by using the QuickChange site-directed mutagenesis kit with the backbone of M-H05-5 （AI-1）, and were named as subgroup A1-2, A2-1, A2-2, BI-1, B1-2, B2-1, and B2-2 respectively. The transient expression of the constructs was investigated by immunofluorescence assay and Western blot analysis. The difference in in cis and in trans NS3 serine protease activity between each subgroup was determined by Western blot analysis. Luciferase reporter assay was used to observe the inhibitory effects of the constructs on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation. Results： The point mutation plasmid constructs were verified for the correct sequence by DNA sequencing. The immunofluorescence assay revealed 4 subcellular localization patterns of NS3, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blot analysis indicated that the incomplete cleavage of NS3/4A appeared in subgroups A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker when compared with the other subgroups. By using NS5A/SBAC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared the other subgroups. Differences in inhibitory effects of HCV NS3 on RIG-I induced IFN-β promoter activity and on p53-dependent transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. Conclusion： The results suggest that subgroup A2-1 and B2-1 has weaker serine protease activities and weaker inhibitory activities on host cell functions than the other subgroups, which might be explained by the different seco

关键词： hepatitis C virus point mutation activity, serine protease secondary structure

Prevalence and genotype distribution of hepatitis B virus among migrant workers in Lombok Island, Indonesia

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Asian Pacific Journal of Tropical Medicine 2020年第1期13卷 8-16页

作者： Laura Navika Yamani Eva Triani Mochamad Amin Juniastuti Takako Utsumi Soetjipto Nasronudin Yoshihiko Yano hak hotta Yoshitake Hayashi Maria Inge Lusida Institute of Tropical Disease Universitas AirlanggaSurabayaEast Java 60115Indonesia Department of Epidemiology Faculty of Public HealthUniversitas AirlanggaSurabayaEast Java 60115Indonesia Indonesia-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases Institute of Tropical DiseaseUniversitas AirlanggaSurabayaEast Java 60115Indonesia Faculty of Medicine Mataram UniversityWest Nusa TenggaraIndonesia Department of Microbiology Faculty of MedicineUniversitas AirlanggaSurabayaEast Java 60131Indonesia Center for Infectious Diseases Kobe University Graduate School of MedicineKobeHyogo 6500017Japan Department of Biochemistry Faculty of MedicineUniversitas AirlanggaSurabayaEast Java 60131Indonesia

Objective:To examine the potential risk of hepatitis B virus(HBV)spread in Indonesia by migrant workers,based on the molecular characteristics of HBV ***:Sera collected from migrant workers traveling to their destination countries(pre-migrant workers)and those returning to Indonesia(post-migrant workers)were screened for HBsAg by ELISA,followed by HBV DNA detection by PCR and(sub)genotype/subtype determination according to surface region and whole genome ***:Of 87 pre-migrant workers,15(17.24%)were HBsAgpositive,whereas 15(12.10%)of 124 post-migrant workers were HBs Ag *** genotype analysis based on the S region showed that HBV-B3/adw2 was predominant(96.15%,25/26)whereas 3.85%(1/26)of isolates were HBV-C3/adrq+.Whole genome sequencing of selected strains and phylogenetic tree analysis identified subgenotype B7 in three samples previously categorized as subgenotype B3 based on S region analysis,supporting a recent argument that subgenotypes B5/B7/B8/B9 could be considered as a quasi-subgenotype of ***:A high prevalence of HBsAg carriers was detected among migrant workers from Lombok Island,with no significant difference in prevalence between before and after returning to *** strains were classified into genotypes common in Indonesia,and the results suggested that migrant workers are not a risk factor for HBV transmission into Indonesia.

关键词： HBV Subgenotypes Migrant workers Indonesia

Molecular epidemiology of hepatitis B virus in Asia

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World Journal of Medical Genetics 2014年第2期4卷 19-26页

作者： Takako Utsumi Yoshihiko Yano hak hotta Indonesia-Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases Institute of Tropical Disease Airlangga University Center for Infectious Diseases Kobe University Graduate School of Medicine

Although safe and effective vaccines against hepatitis B virus(HBV) have been available for three decades, HBV infection remains the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma(HCC) worldwide, especially in Asian countries. HBV has been classified into at least 9 genotypes according to the molecular evolutionary analysis of the genomic DNA sequence and shown to have a distinct geographical distribution. Novel HBV genotypes/subgenotypes have been reported, especially from Southeast Asian countries. The clinical characteristics and therapeutic effectiveness of interferon(IFN) and nucleos(t)ide analogues vary among different HBV genotypes. Mutations at T1653 C in subgenotype C2 from Japan and South Korea, C/A1753 T and C1858 T in subgenotype C1 from Vietnam, and C1638 T and T1753 V in subgenotype B3 from Indonesia were reported to be associated with advanced liver diseases including HCC. Genotype distribution in Japan has been changed by an increasing ratio of subgenotype A2 in chronic hepatitis B. While a large number of epidemiological and clinical studies have been reported from Asian countries, most of the studies were conducted in developed countries such as Taiwan, China, South Korea and Japan. In this review, the most recent publications on the geographical distribution of genetic variants of HBV and related issues such as disease progression and therapy in Asia are updated and summarized.

关键词： Hepatitis B virus Genotype Subgenotype Molecular epidemiology Asia Pathogenicity Drug resistance

Antiviral activity of the dichloromethane extracts from Artocarpus heterophyllus leaves against hepatitis C virus

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Asian Pacific Journal of Tropical Biomedicine 2017年第7期7卷 633-639页

作者： Achmad Fuad Hafid Chie Aoki-Utsubo Adita Ayu Permanasari Myrna Adianti Lydia Tumewu Aty Widyawaruyanti Sri Puji Astuti Wahyuningsih Tutik Sri Wahyuni Maria Inge Lusida Soetjipto hak hotta Institute of Tropical Disease Airlangga University Surabaya 60115 IndonesiaDepartment of Pharmacognosy and Phytochemistry Faculty of Pharmacy Airlangga University Surabaya 60286 Indonesia Department of International Health Kobe University Graduate School of Health Sciences 7-10-2 Tomogaoka Suma-ku Kobe 654-0142 Japan Institute of Tropical Disease Airlangga University Surabaya 60115 Indonesia Department of Biology Faculty of Science and Technology Airlangga University Surabaya 60115 Indonesia Institute of Tropical Disease Airlangga University Surabaya 60115 IndonesiaDepartment of Microbiology Faculty of Medicine Airlangga University Surabaya 60131 Indonesia Institute of Tropical Disease Airlangga University Surabaya 60115 IndonesiaDepartment of Biochemistry Faculty of Medicine Airlangga University Surabaya 60131 Indonesia Department of Oral Vaccine and Drug Development Kobe University Graduate School of Health Sciences 1-5-6 Minatojima-minamimachi Chou-ku Kobe 650-0047 Japan

Objective: To determine anti-viral activities of three Artocarpus species: Artocarpus altilis, Artocarpus camansi, and Artocarpus heterophyllus (A. heterophyllus) against Hepatitis C Virus (HCV). Methods: Antiviral activities of the crude extracts were examined by cell culture method using Huh7it-1 cells and HCV genotype 2a strain JFH1. The mode of action for anti-HCV activities was determined by time-of-addition experiments. The effect on HCV RNA replication and HCV accumulation in cells were analyzed by quantitative reverse transcription-PCR and western blotting, respectively. Results: The dichloromethane (DCM) extract of A. heterophyllus exhibited strong anti-HCV activity with an inhibitory concentration (IC50) value of (1.5 ± 0.6)μg/mL without obvious toxicity. The DCM extracts from Artocarpus altilis and Artocarpus camansi showed moderate anti-HCV activities with IC50 values being (6.5 ± 0.3) μg/mL and (9.7 ± 1.1) μg/mL, respectively. A time-of-addition studies showed that DCM extract from A. heterophyllus inhibited viral entry process though a direct virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by the DCM treatment at high concentration. Conclusions: The DCM extract from A. heterophyllus is a good candidate to develop an antiviral agent to prevent HCV grant reinfection following liver transplantation.

关键词： Hepatitis C virus Artocarpus sp. Artocarpus heterophyllus Antiviral

氨基端不同二级结构的丙型肝炎病毒NS3／4A复合体的丝氨酸酶活性差异研究

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中华医学杂志 2009年第37期89卷 2649-2653页

作者：王雪萍李富军 Lin DENG Motoko NAGANO-FUJII Chunying AN Dapeng JIANG Kikumi KITAYAMA hak hotta 兰州军区兰州总医院中心实验室兰州730050 Department of Microbiology Kobe University Graduate School of Medicine Kobe Japan

目的探讨表达不同氨基端二级结构的丙型肝炎病毒非结构蛋白3／4A（HCVNS3／4A）各亚型丝氨酸酶活性及其对宿主细胞功能抑制作用的差异。方法以pSGS／M。HOS-5／4A为模板（A1-1），构建表达不同氨基端二级结构NS3／4A的点突变质粒，并分... 详细信息

目的探讨表达不同氨基端二级结构的丙型肝炎病毒非结构蛋白3／4A（HCVNS3／4A）各亚型丝氨酸酶活性及其对宿主细胞功能抑制作用的差异。方法以pSGS／M。HOS-5／4A为模板（A1-1），构建表达不同氨基端二级结构NS3／4A的点突变质粒，并分别命名为A1-2，A2-1，A2-2，B1-1，B1-2，B2-1，B2-2。Western印迹检测所有构建质粒的表达及各亚型顺式及反式NS3蛋白酶活性的差异。荧光素酶报告试验检测pSGS／M-H05-5／4A各个二级结构亚型对干扰素-β（IFN-β）产生及p53依赖的荧光素酶基因的转录活性的抑制作用以及各亚型之间抑制作用的差异。结果Western印迹显示所有构建质粒均成功表达，而且A2-1和B2-1亚型NS3／4A存在不完全切割现象。表明与其他亚型相比，A2—1和B2—1顺式NS3丝氨酸蛋白酶活性较弱。与其底物NS5A／SBAC共表达后，A2—1和B2-1亚型未切割NSSASB明显多于其他亚型，而切割NS5A则明显少于其他亚型。说明与其他亚型相比，A2—1和B2．1亚型的反式NS3蛋白酶活性亦较弱。荧光素酶试验结果显示，所有亚型M—H05-5／4A均显著抑制IFN-β启动子活性（P〈0．01）和p53依赖的荧光素酶基因的转录活性（P〈0．01），A2-1和B2-1亚型的抑制作用显著弱于其他亚型（P〈0．05）。结论氨基端不同二级结构的HCVNS3／4A复合体具有不同的丝氨酸酶活性和宿主细胞功能抑制作用。

关键词： C型肝炎样病毒属点突变活性,丝氨酸酶

表达不同氨基端二级结构HCVNS3/4A点突变质粒的构建及在Huh7细胞中的表达

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南方医科大学学报 2009年第4期29卷 720-723,731页

作者：王雪萍李富军长野(藤井)基子北山喜久美堀田博第四军医大学学报编辑部陕西西安710033 兰州军区兰州总医院医务部甘肃兰州730050 神户大学大学院医学系研究科微生物学分野

目的构建表达不同氨基端二级结构HCVNS3/4A的点突变质粒,并在Huh7细胞中进行表达。方法以pSG5/M-H05-5/4A为模板(A1-1),根据分型标准设计点突变引物。首先构建4个单点突变质粒:pSG5/M-H05-5(A1-2)/4A(A1-2)(Y56F),pSG5/M-H05-5(B1-1)/4A(B1-1)(L80Q),pSG5/M-H05-5(B2-1)/4A(B2-1)(V51A),pSG5/M-H05-5(B2-2)/4A(B2-2)(S61A)。然后,分别以A1-2,B2-1,B2-2为模板,再将第80位的赖氨酸点突变为谷氨酸(L80Q),构建另外3个双点突变质粒:pSG5/M-H05-5(B1-2)/4A(B1-2),pSG5/M-H05-5(A2-1)/4A(A2-1)和pSG5/M-H05-5(A2-2)/4A(A2-2)。每个质粒均进行序列测定验证点突变成功。用FuGene6转染试剂将构建物转染入Huh7细胞,并应用间接免疫荧光试验和Western blotting检测构建物的表达。结果免疫荧光实验检测到NS3的4种亚细胞定位方式:点状,弥漫样,面包圈样,及短棒状。Western Blotting亦显示构建物均成功表达,同时发现A2-1和B2-1亚型NS3/4A存在不完全切割现象,表明与其他亚型相比,A2-1和B2-1NS3 in cis丝氨酸蛋白酶活性较弱。结论成功构建表达不同氨基端二级结构HCV NS3/4A点突变质粒,为抗不同亚型HCV的深入研究提供基础。

关键词： pSG5 点突变 HCVNS3/4A 二级结构 Huh 7细胞

pSG5/NS5A5BΔC质粒的构建及在Huh7细胞中的表达

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细胞与分子免疫学杂志 2009年第1期25卷 31-34页

作者：王雪萍李富军邓琳长野(藤井)基子北山喜久美堀田博第四军医大学学报编辑部陕西西安710032 兰州军区兰州总医院医务部甘肃兰州730050 神户大学大学院医学系研究科

目的:构建pSG5/NS5A5B△C质粒,并检验其在Huh7细胞中的瞬时表达。方法:使用已经构建的pTM1-NS2NS5A5B△C质粒为模板,根据pTM1、HCV NS5A5B△C、pSG5序列和酶切位点特点设计引物,PCR扩增得到HCVNS5A5B△C基因片段,将目的片段插入pSG5载体... 详细信息

目的:构建pSG5/NS5A5B△C质粒,并检验其在Huh7细胞中的瞬时表达。方法:使用已经构建的pTM1-NS2NS5A5B△C质粒为模板,根据pTM1、HCV NS5A5B△C、pSG5序列和酶切位点特点设计引物,PCR扩增得到HCVNS5A5B△C基因片段,将目的片段插入pSG5载体中,经筛选阳性克隆、SacⅠ和BglⅡ分别酶切鉴定后,用FuGene6转染试剂转染入Huh7细胞。用免疫荧光染色、Western blot检测HCV NS5A5B△C在Huh-7细胞中的表达。结果:限制性内切酶SacⅠ和BglⅡ分别酶切鉴定后,琼脂糖凝胶电泳结果显示酶切片段大小和插入方向均与预计一致。荧光显微镜下可见转染的Huh7细胞表达带有绿色荧光的HCV NS5A5B△C蛋白,该蛋白主要表达于细胞质,表达率达40%以上。West-ern blot结果也证实在转染pSG5/NS5A5B△C的Huh7细胞中出现了大小与HCV NS5A5B△C(82ku)一致的条带。结论:成功构建了pSG5/NS5A5B△C质粒,并在Huh7细胞中成功瞬时表达,为进一步研究HCV蛋白的功能奠定了基础。

关键词：丙型肝炎病毒非结构蛋白5A5B pSG5 Huh7细胞

Finite-Time Fuzzy Sliding Mode Control for Nonlinear Descriptor Systems

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IEEE/CAA Journal of Automatica Sinica 2021年第6期8卷 1141-1152页

作者： Zhixiong Zhong Xingyi Wang hak-Keung Lam Key Laboratory of Information Processing and Intelligent Control Minjiang UniversityFuzhou 350121China College of Mechanical and Electronic Engineering Fujian Agriculture and Forestry UniversityFuzhou 350108China Department of Engineering King’s College LondonLondonWC2R 2LSUnited Kingdom

This article addresses the finite-time boundedness(FTB)problem for nonlinear descriptor ***,the nonlinear descriptor system is represented by the Takagi-Sugeno(T-S)model,where fuzzy representation is assumed to be appearing not only in both the state and input matrices but also in the derivative *** using a descriptor redundancy approach,the fuzzy representation in the derivative matrix is reformulated into a linear ***,we introduce a fuzzy sliding mode control(FSMC)law,which ensures the finite-time boundedness(FTB)of closed-loop fuzzy control systems over the reaching phase and sliding motion ***,by further employing the descriptor redundancy representation,the sufficient condition for designing FSMC law,which ensures the FTB of the closed-loop control systems over the entire finite-time interval,is derived in terms of linear matrix inequalities(LMIs).Finally,a simulation study with control of a photovoltaic(PV)nonlinear system is given to show the effectiveness of the proposed method.

关键词： Finite-time boundedness(FTB) fuzzy sliding mode control(FSMC) Takagi-Sugeno(T-S)fuzzy descriptor system

Complete Convergence for Randomly Weighted Sums of Random Variables Satisfying Some Moment Inequalities

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Acta Mathematica Sinica,English Series 2021年第2期37卷 279-288页

作者： Ping Yan CHEN Soo hak SUNG Department of Mathematics Ji'nan UniversityGuangzhou 510630P.R.China Department of Applied Mathematics Pai Chai UniversityDaejeon 35345South Korea

For random variables and random weights satisfying Marcinkiewicz-Zygmund and Rosenthal type moment inequalities, we establish complete convergence results for randomly weighted sums of the random variables. Our results generalize those of(Thanh et al. SIAM J. Control Optim., 49,106–124(2011), Han and Xiang J. Ineq. Appl., 2016, 313(2016), Li et al. J. Ineq. Appl., 2017, 182(2017), and Wang et al. Statistics, 52, 503–518(2018).)

关键词： Randomly weighted sum complete convergence moment inequality