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使用ImageJ定量埃博拉病毒感染中细胞间连接传递的方案。

Protocol for quantification of intercellular connection transmission in ebolavirus infections using ImageJ.

作者信息

Santos Rodrigo I, Bukreyev Alexander

机构信息

Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; Galveston National Laboratory, Galveston, TX, USA.

Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; Galveston National Laboratory, Galveston, TX, USA.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103363. doi: 10.1016/j.xpro.2024.103363. Epub 2024 Oct 5.

DOI:10.1016/j.xpro.2024.103363
PMID:39369387
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11491943/
Abstract

Previous work demonstrates that ebolaviruses can spread to neighboring cells through intercellular connections. Here, we present a protocol to quantify the intercellular spread of ebolaviruses via immunofluorescence. We describe steps for cell plating, Bundibugyo virus infection, and adding a neutralizing antibody. We detail procedures for quantitative microscopy assay using ebolavirus immunodetection. Strong virus accumulation around the plasma membrane leads to high fluorescence signal preventing quantification of viral spread based on signal intensity. This protocol minimizes the impact of this bias. For complete details on the use and execution of this protocol, please refer to Santos et al..

摘要

先前的研究表明，埃博拉病毒可通过细胞间连接扩散至邻近细胞。在此，我们提供一种通过免疫荧光定量埃博拉病毒细胞间传播的方法。我们描述了细胞铺板、本迪布焦病毒感染以及添加中和抗体的步骤。我们详细说明了使用埃博拉病毒免疫检测进行定量显微镜分析的程序。质膜周围强烈的病毒聚集导致高荧光信号，从而无法基于信号强度对病毒传播进行定量。本方法可将这种偏差的影响降至最低。有关本方法的使用和执行的完整详细信息，请参考桑托斯等人的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/17975afea1f1/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/2481ba839726/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/8281cb8aa35e/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/1cf7713227e2/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/17975afea1f1/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/2481ba839726/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/8281cb8aa35e/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/1cf7713227e2/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1861/11491943/17975afea1f1/gr3.jpg

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本文引用的文献

1
Blocking of ebolavirus spread through intercellular connections by an MPER-specific antibody depends on BST2/tetherin.通过一种针对 MPER 的抗体阻断埃博拉病毒通过细胞间连接的传播依赖于 BST2/ tetherin。
Cell Rep. 2023 Oct 31;42(10):113254. doi: 10.1016/j.celrep.2023.113254. Epub 2023 Oct 17.
2
Convergence of a common solution for broad ebolavirus neutralization by glycan cap-directed human antibodies.糖萼帽导向的人源抗体对广泛埃博拉病毒中和的共同解决方案的收敛性。
Cell Rep. 2021 Apr 13;35(2):108984. doi: 10.1016/j.celrep.2021.108984.
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Asymmetric antiviral effects of ebolavirus antibodies targeting glycoprotein stem and glycan cap.
针对糖蛋白茎和聚糖帽的埃博拉病毒抗体的非对称抗病毒作用。
PLoS Pathog. 2018 Aug 23;14(8):e1007204. doi: 10.1371/journal.ppat.1007204. eCollection 2018 Aug.
4
Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.多功能 Pan-埃博拉病毒抗体识别埃博拉病毒糖蛋白上的广泛脆弱位点。
Immunity. 2018 Aug 21;49(2):363-374.e10. doi: 10.1016/j.immuni.2018.06.018. Epub 2018 Jul 17.
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Cooperation of the Ebola Virus Proteins VP40 and GP with BST2 To Activate NF-κB Independently of Virus-Like Particle Trapping.埃博拉病毒蛋白VP40和GP与BST2协同作用以独立于病毒样颗粒捕获激活核因子κB
J Virol. 2017 Oct 27;91(22). doi: 10.1128/JVI.01308-17. Print 2017 Nov 15.
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Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.自然感染埃博拉病毒的人类幸存者体内的交叉反应性和强效中和抗体反应
Cell. 2016 Jan 28;164(3):392-405. doi: 10.1016/j.cell.2015.12.022. Epub 2016 Jan 21.
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Cell-cell contact promotes Ebola virus GP-mediated infection.细胞间接触促进埃博拉病毒糖蛋白介导的感染。
Virology. 2016 Jan 15;488:202-15. doi: 10.1016/j.virol.2015.11.019. Epub 2015 Dec 3.
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DENGUE VIRUS. Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers.登革病毒。一种通过锁定E蛋白二聚体来中和2型登革病毒的抗体的冷冻电镜结构。
Science. 2015 Jul 3;349(6243):88-91. doi: 10.1126/science.aaa8651.
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Virus cell-to-cell transmission.病毒细胞间传播。
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10
Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda.新发现的与乌干达出血热疫情相关的埃博拉病毒。
PLoS Pathog. 2008 Nov;4(11):e1000212. doi: 10.1371/journal.ppat.1000212. Epub 2008 Nov 21.