Name: monitoring influenza virus survival outside the host using real-time cell analysis
Uploaded: 2021-02-20T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis at JoVE.com

根据适当的生物安全水平要求（BSL-2或更高，取决于亚型）处理所有流感病毒。使用具有低传代历史（在MDCK细胞上小于5倍）的IAV菌株来确保实验之间的低变异。
1. 试剂和起始材料的制备
<ol><li>MDCK细胞和无菌细胞培养基的制备<ol>
<li>在补充10%热灭活胎儿小腿血清（FCS）和抗生素（100单位/ mL青霉素，100mg / mL链霉素）的改良鹰培养基（MEM）中培养Madin-Darby犬肾（MDCK）细胞。</li>...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+impedance+measurement+as+a+new+tool+for+poxvirus+titration,+antibody+neutralization+testing+and+evaluation+of+antiviral+substances.">Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances.</a> Biochemical and Biophysical Research Communications. 401 (1), 37-41 (2010).</li><li>Lebourgeois, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Real-Time+Cell+Analysis+(RTCA)+Method+as+a+Fast+and+Accurate+Method+for+Detecting+Infectious+Particles+of+the+Adapted+Strain+of+Hepatitis+A+Virus.">Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus.</a> Frontiers in Cellular and Infection Microbiology. 8, 335 (2018).</li><li>Teng, Z., Kuang, X., Wang, J., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+cell+analysis--a+new+method+for+dynamic,+quantitative+measurement+of+infectious+viruses+and+antiserum+neutralizing+activity.">Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.</a> Journal of Virological Methods. 193 (2), 364-370 (2013).</li><li>Fang, Y., Ye, P., Wang, X., Xu, X., Reisen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+flavivirus+induced+cytopathogenesis+using+cell+electric+impedance+technology.">Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology.</a> Journal of Virological Methods. 173 (2), 251-258 (2011).</li><li>Charretier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+real-time+cell+analysis+method+for+determining+viral+infectious+titers+during+development+of+a+viral+vaccine+production+process.">Robust real-time cell analysis method for determining viral infectious titers during development of a viral vaccine production process.</a> Journal of Virological Methods. 252, 57-64 (2018).</li><li>Tian, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Cell+Analysis+for+Measuring+Viral+Cytopathogenesis+and+the+Efficacy+of+Neutralizing+Antibodies+to+the+2009+Influenza+A+(H1N1)+Virus.">Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus.</a> PLoS ONE. 7 (2), 31965 (2012).</li><li>Teng, Z., Kuang, X., Wang, J., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+cell+analysis--a+new+method+for+dynamic,+quantitative+measurement+of+infectious+viruses+and+antiserum+neutralizing+activity.">Real-time cell analysis--a new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity.</a> Journal of Virological Methods. 193 (2), 364-370 (2013).</li><li>Thieulent, C. 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RTCA是一种基于阻抗的技术，越来越多地用于实时监测细胞特性，如细胞粘附、增殖、迁移和细胞毒性。在这项研究中，通过测量病毒失活斜率证明了该技术在宿主外评估IAV存活的能力。TCID50 和斑块测定等挑剔的技术被细胞活力的客观实时评估所取代，从而反映了病毒诱导的细胞病变效应。与TCID50 或斑块形成单元（pfu）类似，CIT50 也与感染的多重性呈线性相关（<strong class...

病毒的传播取决于几个因素的组合。对于环境中分泌的病毒，其传播还取决于在宿主以外的条件下持续存在的能力。因此，一般研究病毒灭活是帮助国家卫生当局和政策制定者实施控制和生物安全措施的关键一步。
在过去十年中，关于病毒在自然环境中和实验室环境中的持久性的知识有了相当大的增加。就甲型流感病毒（IAV）而言，其传播途径使病毒颗粒暴露于各种环境条件。具体而言，它们可以通过1）通过水（即禽类病毒）的粪口途径传播，或2）受污染的污染物以及气溶胶和呼吸道飞沫（即家禽和哺乳动物病毒）直接或间接接触1。在任何情况下，IAV都会受到各种物理化学参数（即pH值，盐度，温度和湿度）的影响，这些参数或多或少会迅速影响其传染性2，3，4，5，6，7，8，9。评估环境因素影响病毒动态以及接触和跨物种传播风险的可能性非常重要，特别是在人畜共患和大流行性病毒方面。
到目前为止，传统的病毒学技术（即通过斑块测定或50%组织培养感染剂量估计测定病毒滴度）已被用于评估IAV感染率随时间推移;但这些技术非常耗时，需要大量耗材10、11、12。使用微电极测量受感染细胞阻抗随时间的变化，是监测不同环境条件下IAV存活率以及一般病毒失活的有用工具。这种方法提供了客观的实时数据，取代了人类对细胞病变效应的主观观察。它可用于确定病毒滴定，从而以较低的置信区间取代传统测量，并避免劳动密集型终点测定。
通过测量细胞阻抗获得的滴定结果与经典斑块测定法或TCID50 方法之间存在线性相关性。因此，通过创建连续稀释病毒的标准曲线，可以使用基于阻抗的滴定方法获得的数据可以很容易地转换为TCID50 或pfu值13，14，15，16，17。使用这种实验方法也可以实现血清样品中存在的中和抗体的检测，定量和功效18，19。最近，基于阻抗的细胞测定已被用于筛选和评估针对Equid甲型疱疹病毒的抗病毒化合物20。
该技术已被用于评估IAV在不同温度下盐水中的持久性，并鉴定IAV血凝素中增加或减少IAV在环境中的持久性的突变21。如果使用传统的滴定方法，这种筛选将需要大量的工作。然而，这种方法可用于任何对细胞形态，细胞数量和细胞表面附着强度有影响的病毒。它还可用于监测各种环境条件下（即在空气中、水中或表面上）的持久性。
此处描述的协议以IAV在水中的生存为例。人类流感病毒在较长时间内暴露于不同的物理化学参数。根据以往结果，选择35 °C盐水（35 g/L NaCl）作为环境模型9。通过细胞感染在不同时间点量化暴露病毒的残留传染性。MDCK细胞是用于IAV扩增的参考细胞类型，接种在涂有微电极传感器的16孔微量滴定板上，并在24小时后被暴露的病毒感染。每15分钟测量一次细胞阻抗，并表示为称为细胞指数（CI）的任意单位。由流感病毒诱导的细胞病变效应，其发病率直接取决于接种到细胞培养物中的感染性病毒颗粒的数量，导致CI降低，随后量化为CIT50 值。该值对应于测量从初始CI减少50%所需的时间（即，在病毒添加之前）。针对多个环境暴露时间计算的 CIT50 值允许在 CIT50 值的线性回归后推断出病毒的失活斜率。

<table>
	<tbody>
		<tr>
			<td>0.25%Trypsin</td>
			<td>ThermoFisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask</td>
			<td>Falcon</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Plate 16 (6 plates)</td>
			<td>ACEA Biosciences, Inc</td>
			<td>5469830001</td>
			<td>E-plates are avalible in different packaging</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life technologies (gibco)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 1X</td>
			<td>Life technologies (gibco)</td>
			<td>31095029</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Life technologies (gibco)</td>
			<td>14040091</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life technologies (gibco)</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr>
			<td>TPCK-Trypsin</td>
			<td>Worthington</td>
			<td>LS003740</td>
			<td></td>
		</tr>
		<tr>
			<td>xCELLigence Real-Time Cell Analysis Instrument S16</td>
			<td>ACEA Biosciences, Inc</td>
			<td>380601310</td>
			<td>The xCELLigence RTCA S16 instruments are available in different formats (16-well, 96-well, single or multi-plate)</td>
		</tr>
	</tbody>
</table>

monitoring influenza virus survival outside the host using real-time cell analysis

病毒颗粒定量方法代表了许多病毒学研究的一个关键方面。尽管存在几种可靠的技术，但它们要么耗时，要么无法检测到微小的变化。这里介绍的是一种通过实时分析受感染细胞的电阻抗变化来精确定量病毒滴度的方案。细胞阻抗是通过位于微孔板中细胞下方的金微电极生物传感器测量的，其大小取决于细胞的数量以及它们的大小和形状。该协议允许以增强的灵敏度实时分析细胞增殖，活力，形态和迁移。还提供了一个实际应用示例，通过量化甲型流感病毒（IAV）的衰变，该病毒随时间推移影响病毒感染性的各种物理化学参数（即温度，盐度和pH值）。对于此类应用，该协议减少了所需的工作量，同时还生成了传染性病毒颗粒的精确定量数据。它允许比较不同IAV之间的失活斜率，这反映了它们在给定环境中的持久化能力。该方案易于执行，具有高度可重复性，可应用于细胞培养中产生细胞病变效应的任何病毒。

使用不同浓度的MDCK细胞（每孔15，000至120，000个细胞）在120小时后获得的原始数据如图 1所示。24小时后，CI测量表明，接种有30，000个细胞的孔中的细胞仍处于生长的指数阶段，并且该细胞浓度用于进一步的实验。 图2 显示了CIT50 值与感染的初始多重性之间的线性相关性。MDCK细胞培养24小时，然后在不同的感染多样性下感染A / Paris / 2590 / 2009 H1N...

Watch this Scientific Journal Video about Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis at JoVE.com

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

这里报道的是使用实时监测受感染细胞的电阻抗来定量感染性病毒颗粒的方案。通过量化甲型流感病毒在模拟环境条件下的不同物理化学参数下的衰变，提出了该方法的实际应用。

使用实时细胞分析监测宿主外的流感病毒存活率

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

各种颗粒定量的方法代表了大多数生物学研究的关键方面。该协议允许实时精确定量病毒数据。这种技术取代了传统的病毒数据测量。通过客观的实时数据，我们低于置信区间，避免了评估劳动密集型终点。该方法可用于诱导细胞病变效应的所有病毒。演示该程序的将是我们实验室的实验室技术人员Quentin Grassin。为了繁殖和扩增H1N1病毒，根据标准方案在两个75平方厘米的组织培养瓶上接种7.5倍10到6个MDCK细胞，并在37摄氏度下孵育细胞24小时，以达到90%至100%汇合度。第二天，用每个烧瓶每次洗涤五毫升无菌PBS洗涤细胞两次，并将一个烧瓶标记为对照。接下来，在装有新鲜病毒传播培养基的1.5毫升管中将解冻的甲型流感病毒原液小瓶稀释至适当的实验浓度。使用一毫升稀释的储备液以1倍10至阴性3的多重感染感染感染MDCK细胞，或1倍10至阴性4个斑块形成单位，并向对照瓶中加入一毫升病毒传播培养基。在室温下将病毒吸附到细胞中45分钟，每15分钟搅拌一次。在孵育结束时，取出接种物并用15毫升病毒传播培养基代替，每毫升补充一微克TPCK胰蛋白酶，以促进病毒血凝素HA0切割成HA1和HA2亚基。然后将烧瓶放入35摄氏度的培养箱中三天。在孵育结束时，在光学显微镜上以40倍物镜比较每个培养物中的细胞，以评估细胞对细胞的细胞病变作用。当细胞病变效应完成时，将细胞培养物上清液加入15毫米管中，并通过离心收集来自每个培养物的细胞碎片。然后，将澄清的病毒培养物上清液转移到15毫升管中，并将后代病毒等分到一次性无菌低温小瓶中以储存零下80摄氏度。为了确定细胞感染的适当细胞量，在用PBS洗涤细胞之前，在37摄氏度下用3毫升0.25%胰蛋白酶-EDTA在3毫升中每烧瓶0.25%的孵育45分钟收获它们之前，按照所示制备约80%汇合的MDCK细胞培养物。当细胞分离时，用每个烧瓶7毫升新鲜培养基停止反应，并计数来自每个培养物的细胞。将细胞稀释至每毫升细胞培养基中第5个细胞的4倍10倍，并按照指示对细胞进行两次连续稀释。将E板在室温下放置几分钟，然后向每个孔中加入100微升细胞培养基，而不接触E板的电极。打开底座，将板前端插入阻抗测量仪的底座口袋。关闭培养箱门并打开软件。在"默认实验模式设置"中，突出显示选定的底座，然后双击顶部以输入实验的名称。单击布局，然后为板的每个选定孔输入必要的样品信息。单击计划、步骤和添加步骤。该软件将自动添加一个一秒钟的步骤来测量背景阻抗，单击执行并开始，继续。单击"绘图并添加"以选择适当的孔，确认背景阻抗介于负 0.1 和 0.1 之间，然后再继续下一步。接下来，从底座中取出板，并将每个细胞悬浮液的100微升一式两份加入适当的孔中。将E板在室温下在层流罩中放置30分钟，以使细胞均匀分布到孔的底部，然后将E板装入摇篮口袋。单击"计划"和"添加步骤"，然后输入值以每 30 分钟监视单元格 200 次重复，然后选择"开始"、"继续"。要检查和绘制细胞阻抗数据，请单击"绘图"并选择接种后24小时静止期之前的细胞浓度，以获得在病毒感染期间仍处于生长阶段的细胞。为了确定CIT50值与感染多重性之间的相关性，在将3次10至第4个新鲜分裂的MDCK细胞培养到电子微量滴定板的每个孔中24小时后，用每孔100微升新鲜病毒繁殖培养基洗涤细胞两次，每次洗涤，并使用单通道移液管向每个孔中加入100微升病毒悬浮液。当所有病毒稀释液都已加入后，在35摄氏度下轻轻地将板装入仪器的底座口袋中，并开始每15分钟监测一次细胞阻抗，至少100小时，如图所示。经过两个周期的测量后，单击以暂停设备，然后从底座中取出E板。向每个孔中加入100微升补充有TPCK胰蛋白酶的病毒传播培养基，并将E板放回摇篮口袋中。然后，开始分析。为了评估甲型流感病毒的生存动力学，在蒸馏水中加入氯化钠至每升35克的终浓度，并将900微升所得盐水溶液加入两毫升冷冻管中。向每个冷冻管中加入100微升病毒储备，并将试管置于35摄氏度的培养箱中，以进行适当的实验时间。在孵育结束的前一天，在16孔微量滴定板中重复步骤，将3倍10至第4个新鲜分裂的MDCK细胞和100微升病毒繁殖培养基的种子重复步骤，允许测量背景阻抗和细胞均匀分布到孔的底部。然后将细胞在37摄氏度和5%二氧化碳下培养24小时。第二天，洗涤细胞两次，然后用在新鲜培养基中稀释10次的100微升盐水蒸馏病毒感染细胞，并每15分钟监测细胞阻抗至少100小时。在这里，显示了使用不同浓度的MDCK细胞在120小时后获得的原始数据。24小时后，细胞指数测量显示，接种3倍10至第4个细胞的孔中的细胞仍处于生长的指数阶段，因此，该细胞浓度用于后续实验。MDCK细胞培养物表明CIT50与流感病毒感染的初始多重性之间存在明显的线性关系。典型的生存动力学实验结果表明，由于病毒诱导的细胞病变效应，细胞指数降低。CIT50可用于计算每种条件下每种病毒的病毒失活斜率，以确定哪种病毒在研究环境中具有最大的稳定性。病毒的稳定性间接地与失活斜率相关，例如，允许鉴定参与甲型流感病毒在宿主外存活的血凝素糖蛋白中的氨基酸残基。这种技术对小的变化非常敏感。因此，鼓励使用自动细胞计数器，以便在实验之间获得可重复的结果。该方法还可用于比较不同病毒的复制，一次研究多个细胞系的病毒向性，或研究病毒周期的特定步骤。

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Real-time Live Imaging of T-cell Signaling Complex Formation

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Influenza Virus Propagation in Embryonated Chicken Eggs

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...