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微生物学报 2022年第2期62卷 672-685页

作者：瞿欢胡靖飞李施倩肖黛张路文付嘉钰赵玉佳陈汭文翼平伍锐赵勤颜其贵文心田曹三杰黄小波四川农业大学动物医学院猪病研究中心四川成都611130 农业农村部兽用药物与兽医诊断技术四川科学观测实验站四川成都611130 四川农业大学国家级动物类实验教学示范中心四川成都611130

【目的】探究猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)能否在不同动物源性细胞系中感染和增殖。【方法】本研究将PDCoV四川分离株CHN-SC2015接种来自仓鼠、家禽、猴、人和猪的12种细胞系,将病毒在每种细胞系中盲传至少5代并通... 详细信息

【目的】探究猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)能否在不同动物源性细胞系中感染和增殖。【方法】本研究将PDCoV四川分离株CHN-SC2015接种来自仓鼠、家禽、猴、人和猪的12种细胞系,将病毒在每种细胞系中盲传至少5代并通过逆转录-聚合酶链式反应(RT-PCR)、实时荧光定量PCR(RT-qPCR)、间接免疫荧光试验(IFA)和测序鉴定。【结果】PDCoV在Vero、PAM、PK15、ST和LLC-PK1细胞中的P1表现出明显的细胞病变效应(CPE),且在PAM、PK15、ST和LLC-PK1细胞上有不同程度的增殖,但在Vero和PAM细胞的后续传代中CPE逐渐消失;除3种易感细胞外(PK15、LLC-PK1和ST),病毒拷贝数随着传代次数的增加而逐渐降低,而在DEF、Marc-145、HEK-293、ZYM-SIEC02和PAM细胞的P4或P5中不能检测到PDCoV;PCR结果显示,只有在CEF和Vero细胞的P5中还能检测到PDCoV;IFA的结果表明,PDCoV可以感染除BHK-21和ZYM-SIEC02以外的大多数细胞,在PK15、LLC-PK1和ST细胞的P1、P3和P9均可以观察到特异性免疫荧光,因此仅3种细胞系(PK15、LLC-PK1和ST)适合病毒的连续传代,在P9病毒滴度分别可达10^(7.11)、10^(7.00)和10^(7.37) TCID_(50)/mL;在不同的细胞系中传代后,PDCoV的S基因序列累计有14个核苷酸和相应的12个氨基酸突变。【结论】本研究表明,PDCoV可在体外感染多种细胞,提示其可能存在跨种传播潜力。

关键词：猪δ冠状病毒细胞系易感性感染跨物种传播

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人氨基肽酶N在猪丁型冠状病毒感染HEK293细胞中的作用

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畜牧兽医学报 2022年第5期53卷 1587-1597页

作者：赵玉佳陈汭宋代丽张路文肖黛李施倩文翼平伍锐赵勤杜森焱颜其贵文心田曹三杰黄小波四川农业大学动物医学院猪病研究中心成都611130 农业农村部兽用药物与兽医诊断技术四川科学观测实验站成都611130 四川农业大学国家级动物类实验教学示范中心成都611130

以HEK293细胞为模型,探讨人氨基肽酶N(human aminopeptidase N,hAPN)在猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)入侵人源细胞中的作用。RT-qPCR/RT-PCR鉴定PDCoV在HEK293细胞上的增殖情况,再用CRISPR/Cas9技术构建敲除hAPN基因的HEK293细胞系,通过CCK-8试验验证敲除细胞和野生型细胞的细胞活性;用RT-qPCR和Western blot检测hAPN敲除和过表达对PDCoV复制的影响;进一步通过同源建模和分子对接预测PDCoV S蛋白和hAPN蛋白的结合位点。结果显示:PDCoV在感染HEK293细胞后12~36 h快速增殖,在36 h达到顶峰,在HEK293细胞上至少可传至2代;hAPN敲除细胞与野生型细胞的细胞活性无明显差异,细胞敲除hAPN可显著抑制PDCoV复制,细胞过表达hAPN可促进PDCoV复制;同源建模和分子对接表明,S1蛋白可与hAPN蛋白结构域II结合,主要是S1蛋白的受体结合基序1(receptor binding motif 1,RBM1)氨基酸残基TYR^(92)、THR^(51)、THR^(48)、PHE^(16)和MET^(14)通过氢键与hAPN蛋白的氨基酸残基PHE^(490)、GLN^(531)、ARG^(528)和SER^(529)结合。hAPN在PDCoV感染人源细胞中发挥重要作用,为深入研究PDCoV的细胞入侵和跨种传播机制提供了新的理论依据。

关键词：猪丁型冠状病毒氨基肽酶N HEK293细胞复制

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Evidence-based guideline for the prevention and management of perioperative infection

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JOURNAL OF EVIDENCE BASED MEDICINE 2023年第1期16卷 50-67页

作者： Wang, Qiaoyu cao, Mingnan Tao, Hua Fei, Zhimin Huang, Xiufeng Liang, Pixia Liu, Baiyun Liu, Jianping Lu, Xiaoyang Ma, Penglin Si, Shuyi Wang, Shuo Zhang, Yuewei Zheng, Yingli Zang, Lei Chen, Xiao Dong, Zhanjun Ge, Weihong Guo, Wei Hu, Xin Huang, Xin Li, Ling Liang, Jianshu Liu, Baoge Liu, Dong Liu, Linna Liu, Songqing Liu, Xianghong Miao, Liyan Ren, Haixia Shi, Guangzhi Shi, luwen Sun, Shumei Tao, Xia Tong, Rongsheng Wang, Cheng Wang, Bin Wang, Jincheng Wang, Jingwen Wang, Xiaoling Wang, Xiaoyan Xie, Jian Xie, Shouxia Yang, Hua Yang, Jianxin You, Chao Zhang, Hongyi Zhang, Yi Zhao, Chengson Zhao, Qingchun Zhu, Jiangguo Ji, Bo Guo, Ruichen Hang, Chunhua Xi, Xiaowei Li, Sheyu Gong, Zhicheng Zhou, Jianxin Wang, Rui Zhao, Zhigang Capital Med Univ Beijing Tiantan Hosp Dept Pharm Beijing Peoples R China Beijing United Family Hosp Dept Pharm Beijing Peoples R China Shanghai Univ Tradit Chinese Med Dept Neurosurg Shuguang Hosp Shanghai Peoples R China Zhejiang Univ Womens Hosp Sch Med Dept Gynecol Hangzhou Peoples R China Capital Med Univ Beijing Tiantan Hosp Dept Neurosurg Beijing Peoples R China Beijing Univ Chinese Med Ctr Evidence Based Med Beijing Peoples R China Zhejiang Univ Affiliated Hosp 1 Sch Med Hangzhou Peoples R China Peking Univ Third Hosp Dept Resp & Crit Care Med Beijing Peoples R China Chinese Acad Med Sci Inst Med Biotechnol Beijing Peoples R China Capital Med Univ Beijing Tiantan Hosp Dept Gen Surg Beijing Peoples R China Chinese Acad Med Sci Fuwai Hosp Dept Pharm Beijing Peoples R China Capital Med Univ Beijing Chao Yang Hosp Dept Orthoped Beijing Peoples R China Sun Yat Sen Univ Affiliated Hosp 1 Dept Pharm Guangzhou Peoples R China Hebei Gen Hosp Dept Pharm Shijiazhuang Peoples R China Nanjing Univ Nanjing Drum Tower Hosp Dept Pharm Affiliated HospMed Sch Nanjing Peoples R China Capital Med Univ Beijing Tiantan Hosp Dept Emergency Beijing Peoples R China Beijing Hosp Dept Pharm Beijing Peoples R China Shandong First Med Univ Shandong Prov Qianfoshan Hosp Dept Pharm Affiliated Hosp 1 Jinan Peoples R China Shanghai Jiao Tong Univ Tongren Hosp Dept Pharm Sch Med Shanghai Peoples R China Capital Med Univ Beijing Tiantan Hosp Dept Nursing Beijing Peoples R China Capital Med Univ Beijing Tiantan Hosp Dept Orthoped Beijing Peoples R China HUST Tongji Hosp Tongji Med Coll Dept Pharm Wuhan Peoples R China Air Force Med Univ Dept Pharm Affiliated Hosp 2 Xian Peoples R China Chongqing Med Univ Dept Gen Surg Affiliated Hosp 3 Chongqing Peoples R China Shandong Univ Dept Pharm Qilu Hosp Jinan Peoples R China Soochow Univ Dept Pharm Affiliated Hosp 1 Suzhou Peoples R China Tianjin First Cent Hosp Dept Pharm Tianjin Peoples R Chi

BackgroundWe have updated the guideline for preventing and managing perioperative infection in China, given the global issues with antimicrobial resistance and the need to optimize antimicrobial usage and improve hospital infection control levels. MethodsWe conducted a comprehensive evaluation of the evidence for prevention and management of perioperative infection, based on the concepts of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. The strength of recommendations was graded and voted using the Delphi method and the nominal group technique. Revisions were made to the guidelines in response to feedback from the experts. ResultsThere were 17 questions prepared, for which 37 recommendations were made. According to the GRADE system, we evaluated the body of evidence for each clinical question. Based on the meta-analysis results, recommendations were graded using the Delphi method to generate useful information. ConclusionsThis guideline provides evidence to perioperative antimicrobial prophylaxis that increased the rational use of prophylactic antimicrobial use, with substantial improvement in the risk-benefit trade-off.

关键词： antimicrobials Delphi GRADE guideline perioperative infection

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不同rs-fMRI数据处理方法在血管性认知障碍中的应用

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康复学报 2019年第1期29卷 70-74页

作者：关莹朱路文王丰曹丹娜李晓陵唐强黑龙江中医药大学黑龙江哈尔滨150040 黑龙江中医药大学附属第二医院黑龙江哈尔滨150001 黑龙江中医药大学附属第一医院黑龙江哈尔滨150040

血管性认知障碍(VCI)是指由长期高血压、糖尿病、高血脂等脑血管危险因素导致的与小血管损伤有关的痴呆。随着人口老龄化的加速,VCI发病率呈现出逐年增高的趋势,约40%的脑血管病患者合并认知障碍,表现为不同程度的记忆、语言、视空间功... 详细信息

血管性认知障碍(VCI)是指由长期高血压、糖尿病、高血脂等脑血管危险因素导致的与小血管损伤有关的痴呆。随着人口老龄化的加速,VCI发病率呈现出逐年增高的趋势,约40%的脑血管病患者合并认知障碍,表现为不同程度的记忆、语言、视空间功能障碍,严重影响患者日常生活活动、社交活动及工作能力。VCI已成为仅次于阿尔茨海默病(AD)的第二大痴呆类型,给社会及家庭带来了沉重的负担。如果能早期发现并及时治疗,可有效改善VCI患者的预后。静息态功能磁共振(rs-fMRI)是检测受试者于安静、平卧、无运动,闭眼或睁眼,但未进入睡眠,无系统思维活动的静息状态下不同脑区的自发性神经元活动,建立各相关脑区之间的网络与功能连接,反映静息状态下大脑自发的功能活动。广义rs-fMRI包括血氧水平依赖功能磁共振成像、磁共振波谱、磁敏感加权成像及弥散张量成像等;狭义rs-fMRI特指血氧水平依赖功能磁共振成像,该技术主要是通过反映大脑的血氧水平依赖信号的自发活动来反映神经元的自发性活动,相较于任务态功能磁共振更接近生理状态。作为一种敏感的辅助检查手段,rs-fMRI以其独特的优势已广泛应用于VCI的神经机制及诊疗研究。在临床应用中,针对VCI的rs-fMRI数据处理方法很多,如低频振荡振幅(ALFF)、局部一致性(ReHo)、功能连接(FC)、脑网络等。不同的数据处理方法有其不同的意义、优缺点及应用价值,且不同的数据处理和分析方法对结果又存在一定的差异,如ALFF、ReHo属于局部功能活动特性分析方法;FC和脑网络则属于线性相关分析方法。本文就这几种数据处理方法梳理了近年来关于rs-fMRI在VCI领域中的研究,归纳了常用的rs-fMRI数据处理方法、特征,综合论述了rs-fMRI不同数据处理方法在VCI中的应用,旨在为VCI的临床诊断及治疗提供影像学依据。

关键词：血管性认知障碍静息态功能磁共振低频振荡振幅局部一致性功能连接脑网络

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Interferon regulatory factor 7 is involved in the growth of Epstein Barr virus-transformed human B lymphocytes

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VIRUS RESEARCH 2015年 195卷 112-118页

作者： Xu, Dongsheng Zhang, Yanyan Zhao, Lingjun cao, Mingxia Lingel, Amy Zhang, luwen Tongji Univ Sch Med Shanghai Peoples Hosp 10 Ctr Neuroimmunol & Regenerat Therapy Shanghai 200072 Peoples R China Wuhan Univ Coll Life Sci State Key Lab Virol Wuhan 430072 Peoples R China Univ Nebraska Sch Biol Sci Nebraska Ctr Virol Lincoln NE 68588 USA St Louis Univ Hlth Sci Ctr Inst Mol Virol St Louis MO 63104 USA

The cellular interferon (IFN) regulatory factor-7 (IRF7) is closely associated with the Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes in vitro and in vivo. However, the exact role of IRF7 in viral transformation is not clear. We have found that knockdown of IRF7 leads to growth inhibition Of EBV-transformed cells, and restoration of IRF7 by exogenous plasmid correlates with growth recovery of the viral transformed cells. In addition, IRF7-knockdown cells have a lower proliferation but a higher apoptotic rate than control cells. Moreover, reduction of IRF7 leads to reduction of major viral oncoprotein, latent membrane protein 1 (LMP1). Our data suggest that IRF7 may be a factor in EBV transformation and a useful target in the therapy of EBV-mediated neoplasia. (C) 2014 Elsevier B.V. All rights reserved.

关键词： IRF7 EBV Lymphoma Transformation LMP1

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Nuclear factor κB represses the expression of latent membrane protein 1 in Epstein-Barr virus transformed cells

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World Journal of Virology 2014年第4期3卷 22-29页

作者： Mingxia cao Qianli Wang Amy Lingel luwen Zhang School of Biological Sciences University of NebraskaLincolnNE 68588United States 238 Morrison Center School of Biological SciencesNebraska Center for VirologyUniversity of NebraskaLincolnNE 68583-0900United States

AIM: To investigate the role of nuclear factor κB(NF-κB) in the regulation of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) in EBV transformed cells. METHODS: LMP1 expression was examined in EBV transformed human B lymphocytes with modulation of NF-κB activity. RESULTS: EBV infection is associated with several human cancers. EBV LMP1 is required for efficient transformation of adult primary B cells in vitro, and is expressed in several pathogenic stages of EBVassociated cancers. Regulation of EBV LMP1 involves both viral and cellular factors. LMP1 activates NF-κB signaling pathway that is a part of the EBV transformation program. However, the relation between NF-κB and LMP1 expression is not well established yet. In this report, we found that blocking the NF-κB activity by Inhibitor of κB stimulated LMP1 expression, while the overexpression of NF-κB repressed LMP1 expression in EBV-transformed IB4 cells. In addition, LMP1 repressed its own promoter activities in reporter assays, and the repression was associated with the activation of NF-κB. Moreover, NF-κB alone is sufficient to repress LMP1 promoter activities. CONCLUSION: Our data suggest LMP1 may repress its own expression through NF-κB in EBV transformed cells and shed a light on LMP1 regulation during EBV transformation.

关键词： Nuclear factorκB Epstein-Barr virus Latent membrane protein 1 Latency Transformation

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TLR-TRIF Pathway Enhances the Expression of KSHV Replication and Transcription Activator

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JOURNAL OF BIOLOGICAL CHEMISTRY 2013年第28期288卷 20435-20442页

作者： Meyer, Florencia Ehlers, Erica Steadman, Andrew Waterbury, Thomas cao, Mingxia Zhang, luwen Univ Nebraska Sch Biol Sci Lincoln NE 68583 USA Univ Nebraska Nebraska Ctr Virol Lincoln NE 68583 USA

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gamma-herpesvirus. KSHV replication and transcription activator (RTA) is necessary and sufficient for KSHV reactivation from latency. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, act through adaptors, and initiate innate and adaptive immune responses against pathogens. Toll/interleukin-1-receptor domain containing adaptor protein inducing interferon-beta (TRIF) is an adaptor associated with TLR3 and TLR4 signaling, and is closely related to antiviral signaling to activate type I interferon (IFN) production. We previously found that KSHVRTA degrades TRIF indirectly and blocks TLR3 pathways. In this report, we find that TRIF, as well as TLR3 activation, enhances KSHVRTA protein expression. The C-terminal region of the RTA is involved in the responding TRIF-mediated enhancement. The degradation of TRIF and the enhancement of RTA expression are using two different pathways. The enhancement by TLR-TRIF is at least partially via promoting translational efficiency of RTA mRNA. Finally, the receptor-interacting protein 1 (RIP1) may be involved in TRIF-mediated enhancement of RTA expression, but not in the RTA-mediated degradation of TRIF. Therefore, the activation of TLR-TRIF pathway enhances KSHV RTA protein expression, and KSHV RTA in turn degrades TRIF to block innate immunity. The putative KSHV-TLR-adaptor-interacting loop may be a critical element to evade and usurp host innate immunity in KSHV life-cycle.

关键词： Innate Immunity Toll-like Receptors (TLR) Translation Control TRIF Tumor Viruses

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APOBEC3G真核表达载体的构建及其对HBV复制表达的影响

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武汉大学学报（医学版） 2009年第2期30卷 200-204页

作者：王鲁文楮小刚刘艳红曹廷华龚作炯武汉大学人民医院感染科/武汉大学病毒学国家重点实验室湖北武汉430060

目的:构建APOBEC3G真核表达载体,在体外初步探讨APOBEC3G对HBV复制表达的影响。方法:应用RT-PCR法从人PBMC细胞中扩增APOBEC3G基因,分子克隆法构建表达APOBEC3G蛋白的真核表达载体pcDNA3.1-APOBEC3G,并脂质体法转染HepG22.2.15细胞。Wes... 详细信息

目的:构建APOBEC3G真核表达载体,在体外初步探讨APOBEC3G对HBV复制表达的影响。方法:应用RT-PCR法从人PBMC细胞中扩增APOBEC3G基因,分子克隆法构建表达APOBEC3G蛋白的真核表达载体pcDNA3.1-APOBEC3G,并脂质体法转染HepG22.2.15细胞。Western-blot鉴定细胞中APOBEC3G蛋白的表达。ELISA法检测细胞培养上清液中HBsAg和HBeAg水平,实时定量PCR检测HBVDNA和HBV mRNA水平。结果:经酶切鉴定及测序证实APOBEC3G真核表达载体被成功构建,APOBEC3G蛋白可在HepG22.2.15细胞中表达。转染APOBEC3G重组质粒的HepG22.2.15细胞的HBsAg、HBeAg、HBVDNA及HBV mRNA水平均明显低于未转染重组质粒的HepG22.2.15细胞。结论:APOBEC3G明显抑制了HepG22.2.15细胞中HBV的复制与表达,成功构建APOBEC3G真核表达载体,为深入研究APOBEC3G抗HBV的作用机制奠定实验基础。

关键词： APOBEC3G 基因构建真核表达乙型肝炎病毒 HepG22.2.1 5细胞

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