Virological and Immunological Characteristics of Human Echovirus 25 and a Murine Model of Coxsackievirus A16 Infection

由人肠道病毒感染引起的手足口病（Hand,FootandMouthDisease，HFMD）多发生于婴幼儿，在我国法定丙类传染性疾病中发病率和死亡率最高，严重危害婴幼儿的生命和健康。2014年我国手足口病的发病率超过2012年和2013年同期的发病率，当前尚无投入实际应用的疫苗或特效药物。目前我国大陆地区的HFMD疫情主要与肠道病毒71型（EV71）和柯萨奇病毒A16型（CA16）有关，但由于肠道病毒种类多样，一些较少被关注的肠道病毒同样也可导致较为严重的疫情，近期在美国发生的肠道病毒D68型（EV-D68）疫情就是一个典型的案例。因此，开展相关的病原体流行病学监控及对不同肠道病毒的基础性研究...Hand, foot and mouth disease (HFMD) is a common infectious disease in infants caused by enterovirus, with high morbidity and mortality in Class-C communicable disease. In 2014, the incidence of HFMD remains higher than 2012 and 2013. Currently, effective chemoprophylaxis or vaccination approaches for dealing with HFMD are still not available. The most common strains causing HFMD are coxsackievirus...学位：理学博士院系专业：生命科学学院_生物化学与分子生物学学号：2172008015042

Development and Application of a Novel Neutralization Test for Echovirus 25

目的:建立一种新型的快速、高通量的埃可病毒25型(ECHO25)中和抗体检测方法,并初步评价其在ECHO25中和抗体筛选和血清流行病学调查中的应用价值。方法:应用免疫荧光方法筛选ECHO25高亲和性抗体并将其作为检测单抗,结合酶联免疫斑点检测技术(ELISPOT)建立ECHO25中和抗体检测方法;使用不同效价的血清评价该方法的准确性;采用所建立的中和方法对ECHO25单克隆抗体、临床血清样品进行检测。结果:建立了快速检测ECHO25中和抗体的Nt-ELISPOT方法,以ECHO25单克隆抗体5B9作为检测抗体;相比经典的中和实验方法 Nt-CPE,该方法可显著缩短检测时间(从5~7 d缩短至1 d以内),检测结果具有较好的一致性;采用所建立的Nt-ELISPOT方法首次筛选获得3株对ECHO25具有较好中和能力的单克隆抗体;临床血清样品检测结果显示厦门地区可能存在ECHO25的流行。结论:该方法可以应用于中和抗体筛选和血清学的临床辅助诊断,为ECHO25的防治研究提供支持。Objective: To establish a rapid and high-throughput neutralization test for echovirus 25(ECHO25),and evaluate its application in neutralizing antibody screening and seroepidemiological surveys. Methods: Immuno-fluorescence assay was applied to screen a high affinity antibody, which was used as the detection antibody forECHO25, and a rapid neutralization test was established based on enzyme- linked immunospot assay(Nt-ELISPOT). The accuracy of this method was evaluated by detecting serum samples with different titer. Monoclonalantibodies against ECHO25 and clinical serum samples were detected via the established neutralization test. Results: A rapid method to detect neutralizing antibody against ECHO25 was established and an anti-ECHO25 anti-body, 5B9, was used as the detection antibody. The detection period could be shortened significantly comparedwith the classical neutralization test(Nt- CPE)(from five to seven days to less than one day), and the Nt-ELISPOT had good consistency with the Nt- CPE. Meanwhile, three neutralizing antibodies for ECHO25 werescreened firstly by this method. The detection results of clinical serum samples showed that infection of ECHO25 might be popular in Xiamen. Conclusion: This method can be used in neutralizing antibody screening and seroepi-demiological surveys, and it may provide support for the control of ECHO25.国家自然科学基金(81371817,81401669

一种可药物筛选及体内检测的新型慢病毒基因治疗载体的构建

为实现基因治疗过程中的有效药物筛选及体内检测,首次利用核糖体内部进入位点(IRES)构建了同时携带O6-烷基鸟嘌呤-DNA烷基转移酶(MGMT)的突变型P140K基因和荧光素酶(Luciferase)基因的慢病毒载体pBobi-MIL。RT-PCR、免疫荧光、药物筛选克隆形成及化学发光检测等实验结果表明感染重组慢病毒L-MIL的细胞能同时表达MGMT及Luciferase。构建成功的新型慢病毒载体为今后的基因治疗奠定了基础,也为慢病毒滴度的确定提供了一种新的可能

Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

新型冠状病毒SARS-CoV-2在全球蔓延，给全球公共卫生带来严重威胁。快速研制疫苗、抗体和治疗药物成为科学界面临的重大挑战。由于SARS-CoV-2的高度传染性，采用病毒感染模型进行中和抗体及小分子抑制剂的药效评估需要在高等级生物安全实验室中进行，且常需要数天时间才能完成检测，限制了抗体和药物筛选的效率。发展快速、可视、不依赖于活病毒的新冠病毒入胞检测探针和细胞模型，对于加速新冠病毒抗体和药物的研究有重要意义。夏宁邵教授团队通过CHO真核表达系统高效表达制备出C端融合抗酸荧光蛋白Gamillus的重组新冠病毒spike蛋白STG。STG经SEC分子筛和冷冻电镜确认呈现与天然病毒刺突高度相似的三聚体结构，且与ACE2有很高的亲和力（18.2nM）。STG具备良好的细胞相容性和荧光性质，研究者进一步开发了可定量测定感染恢复期血清、疫苗免疫血清中和抗体（入胞阻断抗体）水平的CSBT检测方法。除了抗体检测评估方面的应用外，该研究发展的探针和模型还可用于筛选分析抑制新冠病毒入胞及胞内转运的小分子化合物。 我校博士后张雅丽，博士生王邵娟、巫洋涛，博士后侯汪衡、袁伦志和深圳市第三人民医院沈晨光博士为共同第一作者。厦门大学夏宁邵教授、袁权教授、程通教授为该论文共同通讯作者。The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system,a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed.In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.This study was supported by National Natural Science Foundation of China (81993149041 for N.X.; 81902057 for Y.Z.; 81871316 and U1905205 for Q.Y.), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402‐002‐003 for T.C. and No. 2017ZX10202203‐009 for Q.Y.), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003‐005‐003 for T.C.), the Science and Technology Major Project of Fujian (2020YZ014001), the Science and Technology Major Project of Xiamen (3502Z2020YJ01), and the Guangdong Basic and Applied Basic Research Foundation (2020A1515010368 for C.S.). 该研究得到了国家自然科学基金、传染病防治国家科技重大专项、福建省应急科技攻关项目和厦门应急科技攻关项目的支持

Development of a Neutralization Test for Enterovirus D68 Using the Enzyme-linked Immunospot Assay

针对肠道病毒68型(EV-D68)建立一种新型快速、高效的中和试验方法,并初步评价其在EV-D68流行病学研究中的应用价值。以灭活EV-D68病; 毒免疫Balb/c小鼠,利用杂交瘤技术制备其单克隆抗体;筛选反应性好、特异性强的抗EV-D68单克隆抗体作为检测抗体,基于酶联免疫斑点检测技术(; ELISPOT)建立EV-D68高效中和试验方法;进一步比较该方法与传统中和试验方法Nt-CPE的一致性,并使用该方法对临床血清样本进行检测。共; 获得10株分泌抗EV-D68单克隆抗体的杂交瘤细胞株,选取一株性能最优的15C5进行生产纯化,鉴定其为IgG3亚型;以15C5为检测抗体,建立了; 快速检测EV-D68中和抗体的Nt-ELISPOT方法;与Nt-CPE方法比较,Nt-ELIS-POT方法将检测时间由5~7d缩短至ld以内,并; 且两种方法检测结果一致性良好;应用所建立的Nt-ELISPOT方法对厦门地区146份人群血清样本进行检测,显示血清样本EV-D68中和抗体阳性率; 为98. 6%; (144/146),提示厦门地区可能存在过EV-D68的流行。本研究基于ELISPOT建立的新型EV-D68中和试验方法快速可靠,适合通量检测,; 可以应用于EV-D68中和抗体水平的血清学调查,为EV-D68感染的防控工作提供帮助。We wished to establish a novel, rapid and efficient neutralization test; for enterovirus D68 (EV-D68) and evaluate its application in; seroepidemiological studies. Balb/c mice were immunized with inactivated; EV-D68. Monoclonal antibodies against EV-D68 were prepared by the; hybridoma method. Anti-EV-D68 monoclonal antibodies with high affinity; and specificity were selected as detection antibodies for the; establishment of a rapid neutralization test for EV-D68 using the; enzyme-linked immunospot (ELISPOT) assay. Also, the consistency between; the established method and conventional one, neutralization test based; on the inhibition of cytopathic effects (Nt-CPE),was investigated.; Finally, the established method was evaluated with clinical serum; samples. Ten hybridoma cell lines secreting monoclonal antibodies; against EV-D68 were obtained, and the best of them, 15C5, was selected; for the production and characterization of monoclonal antibodies.; Results showed that 15C5 was an immunoglobulin(Ig) G3 subclass. A rapid; method for the detection of neutralizing antibodies against EV-D68,; Nt-ELISPOT, was established using 15C5 as the detection antibody.; Nt-ELISPOT could shorten the detection time substantially from 5~7 days; in conventional Nt-CPE to <1 day, and there was good consistency between; the two methods. The established Nt-ELISPOT was applied to 146 clinical; serum samples from Xiamen, China, and the positive detection rate was; 98. 6% (144/146),suggesting that EV-D68 infection might be prevalent in; Xiamen. The established novel neutralization test for EV-D68 based on; ELISPOT was rapid and reliable. This method could be used to detect; EV-D68 neutralizing antibodies in seroepidemiological studies, thereby; providing support for the prevention and control of EV-D68 infection.国家自然科学基金; 国家自然科学基金; 福建省自然科学基

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies