Name: virus propagation and cell-based colorimetric quantification
Uploaded: 2023-04-07T14:55:00
Description: Watch this Scientific Journal Video about Virus Propagation and Cell-Based Colorimetric Quantification at JoVE.com

这项研究得到了马来西亚高等教育部的长期研究资助计划（LRGS MRUN Phase 1： LRGS MRUN/F1/01/2018）的支持，并为高等院校卓越中心（HICoE）计划（MO002-2019）提供资金。本研究中的图 3 显示了病灶形成测定的染色工作流程，改编自 BioRender.com （2022） 的“DAB 免疫组织化学”。取自 https://app.biorender.com/biorender-templates/t-5f3edb2eb20ace00af8faed9-dab-immunohistochemistry。

1. 病毒传播
<ol><li>细胞制备<ol><li>在含有 12 mL Dulbecco 改良 Eagle 培养基 （DMEM） 的 75 cm2 细胞培养瓶中培养 Vero 细胞，补充有 10% 胎牛血清 （FBS） 和 2 mM L-谷氨酰胺（参见 材料表）。在37°C的细胞培养箱中用5%CO2孵育细胞。</li>
		<li>在显微镜下监测细胞;一旦细胞达到70%-90%的汇合度，它们就可以使用了（图1A）。 注意：Vero细胞大约?...

<ol>
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R., 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=Zika+virus+in+the+Americas:+early+epidemiological+and+genetic+findings.">Zika virus in the Americas: early epidemiological and genetic findings.</a> Science. 352 (6283), 345-349 (2016).</li><li>Rapid risk assessment: Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barr&#233; syndrome - 4th update. 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S., Niedrig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+yellow+fever+virus:+a+comparison+of+quantitative+real-time+PCR+and+plaque+assay.">Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.</a> Journal of Virological Methods. 110 (2), 185-191 (2003).</li><li>Dulbecco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+plaques+in+monolayer+tissue+cultures+by+single+particles+of+an+animal+virus.">Production of plaques in monolayer tissue cultures by single particles of an animal virus.</a> Proceedings of the National Academy of Sciences. 38 (8), 747-752 (1952).</li><li>Nahapetian, A. T., Thomas, J. N., Thilly, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+environment+for+high+density+Vero+cell+culture:+effect+of+dissolved+oxygen+and+nutrient+supply+on+cell+growth+and+changes+in+metabolites.">Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites.</a> Journal of Cell Science. 81, 65-103 (1986).</li><li>Agbulos, D. S., Barelli, L., Giordano, B. V., Hunter, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zika+virus:+quantification,+propagation,+detection,+and+storage.">Zika virus: quantification, propagation, detection, and storage.</a> Current Protocols in Microbiology. 43, (2016).</li><li>Brien, J. D., Lazear, H. M., Diamond, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+quantification,+detection,+and+storage+of+West+Nile+virus.">Propagation, quantification, detection, and storage of West Nile virus.</a> Current Protocols in Microbiology. 31, (2013).</li><li>Bol&#237;var-Marin, S., Bosch, I., Narv&#225;ez, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+the+focus-forming+assay+and+digital+automated+imaging+analysis+for+the+detection+of+dengue+and+Zika+viral+loads+in+cultures+and+acute+disease.">Combination of the focus-forming assay and digital automated imaging analysis for the detection of dengue and Zika viral loads in cultures and acute disease.</a> Journal of Tropical Medicine. 2022, 2177183 (2022).</li><li>Dangsagul, W., 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=Zika+virus+isolation,+propagation,+and+quantification+using+multiple+methods.">Zika virus isolation, propagation, and quantification using multiple methods.</a> PLoS One. 16 (7), e0255314 (2021).</li><li>Brien, J. 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=Genotype-specific+neutralization+and+protection+by+antibodies+against+dengue+virus+type+3.">Genotype-specific neutralization and protection by antibodies against dengue virus type 3.</a> Journal of Virology. 84 (20), 10630-10643 (2010).</li><li>Lazear, H. M., 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=A+mouse+model+of+Zika+virus+pathogenesis.">A mouse model of Zika virus pathogenesis.</a> Cell Host &amp; Microbe. 19 (5), 720-730 (2016).</li><li>Miner, J. J., 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=Zika+virus+infection+during+pregnancy+in+mice+causes+placental+damage+and+fetal+demise.">Zika virus infection during pregnancy in mice causes placental damage and fetal demise.</a> Cell. 165 (5), 1081-1091 (2016).</li><li>Kang, W., Shin, E. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+focus-forming+assay+with+automated+focus+counting+by+image+analysis+for+quantification+of+infectious+hepatitis+C+virions.">Colorimetric focus-forming assay with automated focus counting by image analysis for quantification of infectious hepatitis C virions.</a> PLoS One. 7 (8), e43960 (2012).</li><li>Brien, J. 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有几种测定方法可以确定病毒滴度;PFA 具有与 FFA 相似的病毒定量方案，其中病毒接种物被稀释以区分单个斑块或病灶。染色后，每个斑块或病灶指示接种物19中的单个感染性颗粒。PFA用结晶紫染色，以可视化由细胞裂解或死亡引起的斑块形成。因此，PFA更耗时，因为病毒引起CPE需要更长的时间，并且仅限于诱导细胞裂解或死亡的病毒。许多实验室已成功使用 FFA 测定黄病毒的传?...

寨卡病毒（ZIKV）感染是一种新出现的蚊媒病毒性疾病。1947 年在乌干达首次分离出 ZIKV 1,2;从 1947 年到 2007 年，它仍然被忽视，因为临床症状最常见的是无症状的，其特征是自限性发热性疾病。2007 年，寨卡病毒流行始于雅浦群岛 3,4，随后于 2013 年至 2014 年在太平洋地区（法属波利尼西亚、复活节岛、库克群岛和新喀里多尼亚）发生更大规模的流行病 5,6,7,8，其中首次在成人中报告了严重的神经系统并发症吉兰-巴雷综合征 （GBS）9.在2015年和2016年期间，早在2013年巴西出现亚洲寨卡病毒基因型后，寨卡病毒首次大范围流行席卷美洲10。在这次疫情期间，新生儿中报告了44万至130万例小头畸形和其他神经系统疾病病例11。目前尚无针对寨卡病毒感染的特异性治愈或治疗方法;因此，迫切需要能够预防感染的寨卡病毒疫苗，特别是在怀孕期间。
病毒定量是确定样品中存在的病毒数量的过程。它在研究中发挥着重要作用，学术实验室涉及许多领域，例如医学和生命科学。这一过程在商业领域也很重要，例如病毒疫苗、重组蛋白、病毒抗原或抗病毒药物的生产。许多方法或检测方法可用于病毒定量12。方法或检测方法的选择通常取决于病毒特征、所需的准确度以及实验或研究的性质。通常，定量病毒的方法可分为两类：检测病毒核酸（DNA或RNA）存在的分子测定和体外测量病毒感染性的测定12。定量聚合酶链反应（qPCR，用于DNA）或定量逆转录聚合酶链反应（qRT-PCR，用于RNA）13和数字液滴PCR14是用于定量给定样品中病毒核酸的常用分子技术的例子15。然而，这些高度灵敏的分子技术无法区分活病毒和非活病毒15。因此，使用上述分子技术无法完成需要生物学特征信息的研究，例如病毒对细胞的感染性;需要能够测量和确定活病毒存在的检测方法。测量病毒传染性的检测方法包括斑块形成试验 （PFA）、50% 组织培养感染剂量 （TCID50）、荧光焦点试验和透射电子显微镜 （TEM）12。
PFA 由 Renato Dulbecco 于 1952 年开发，是最常用的病毒滴定方法之一，包括 ZIKV16。它用于直接测定传染性裂解病毒粒子的病毒浓度。该方法基于裂解病毒在病毒感染后在接种的细胞单层中产生细胞病变效应（CPEs;细胞死亡区或斑块，被未感染细胞包围的感染区域）的能力。然而，该测定存在一些影响其效用的缺点。该检测非常耗时（大约需要 7-10 天，具体取决于病毒），依赖于 CPE，并且容易出错。在本研究中，我们报告了一种免疫比色技术，即焦点形成测定 （FFA），用于检测和定量 24 孔板和 96 孔板形式的 ZIKV。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Polyethersulfone syringe filter</td>
			<td>Sartorius</td>
			<td>S6534-FMOSK</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Nest</td>
			<td>615601</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL sterile serological pipette</td>
			<td>Labserv</td>
			<td>14955156</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Dulbecco&#8217;s phosphate-buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL Screw cap tube&#160;</td>
			<td>Axygen</td>
			<td>SCT-200-SS-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Sterile serological pipettes</td>
			<td>Labserv</td>
			<td>14955157</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;Diaminobenzidine (DAB) peroxidase substrate</td>
			<td>Thermo Scientific</td>
			<td>34065</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C incubator with 5% CO2</td>
			<td>Sanyo</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL sterile serological pipette</td>
			<td>Labserv</td>
			<td>14955155</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>LAB352070</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask&#160;</td>
			<td>Corning</td>
			<td>430725U</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-flavivirus monoclonal antibody, 4G2 (clone D1-4G2-4-15)</td>
			<td>MilliporeSigma</td>
			<td>MAB10216</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved 20x Phosphate buffered saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>22.8 g of 8 mM Na2HPO4, 4.0 g of 1.5 mM KH2PO4, 160 g of 0.14 M NaCl, 4.0 g of 2.7 mM KCl, 1 L of MilliQ H2O</td>
		</tr>
		<tr>
			<td>Biological safety cabinet, Class II</td>
			<td>Holten</td>
			<td>HB2448</td>
			<td></td>
		</tr>
		<tr>
			<td>CTL S6 Universal ELISpot/FluoroSpot Analyzer</td>
			<td>ImmunoSpot, Cellular Technology Limited (CTL)</td>
			<td>CTL-S6UNV12</td>
			<td>Commercial software analyzer</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Bovogen</td>
			<td>SFBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP)</td>
			<td>MilliporeSigma</td>
			<td>12-349</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Laboroptik LTD</td>
			<td>Neubauer improved</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 detergent</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td>Octylphenoxy poly(ethyleneoxy)ethanolIGEPAL&#160;</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>ZEISS</td>
			<td>TELAVAL 31</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory rocker</td>
			<td>FINEPCR</td>
			<td>CR300</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Low viscosity carboxymethyl cellulose (CMC)</td>
			<td>Sigma-Aldrich</td>
			<td>C5678</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette (10 - 100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3125000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette (30 - 300 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3125000052</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (10 - 100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000047</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (100 - 1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (20 - 200 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>Sunlac Low Fat</td>
			<td>N/A</td>
			<td>Prepare 3% Skim milk in 1x PBS for blocking stage in staining</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>Clorox</td>
			<td>N/A</td>
			<td>To disinfect any discarded infectious liquid waste from flasks/plates</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe disposable, Luer Lock, 10 mL with 21 G Needle</td>
			<td>Terumo</td>
			<td>SS10L21G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero African green monkey kidney cells&#160;</td>
			<td>-</td>
			<td>ECACC 88020401</td>
			<td>Received from collaborator. However, Vero cells obtained from other suppliers should be able to be used with some optimization.</td>
		</tr>
	</tbody>
</table>

virus propagation and cell-based colorimetric quantification

寨卡病毒（ZIKV）是一种蚊媒病毒，属于 黄病毒属。ZIKV感染与先天性脑部异常有关，并可能与成人的吉兰-巴雷综合征有关。研究寨卡病毒以了解疾病机制对于促进疫苗和治疗开发非常重要。在病毒学领域，量化病毒的方法至关重要。焦点形成测定 （FFA） 是一种病毒定量测定，它使用抗体检测病毒抗原并使用过氧化物酶免疫染色技术鉴定细胞的感染病灶。目前的研究描述了使用 24 孔和 96 孔（高通量）格式的病毒传播和定量方案。与其他类似研究相比，该协议进一步描述了病灶大小优化，可以作为扩大该测定对其他病毒使用范围的指南。首先，ZIKV在Vero细胞中繁殖3天。使用FFA收获并定量含有ZIKV的培养上清液。简而言之，将病毒培养物接种到Vero细胞上并孵育2-3天。然后，在优化的染色过程（包括细胞固定、透化、封闭、抗体结合和与过氧化物酶底物孵育）后确定病灶形成。使用体视显微镜（24孔格式的手动计数）或软件分析仪（96孔格式的自动计数）观察染色的病毒病灶。FFA 提供可重复的、相对较快的结果（3-4 天），适用于不同的病毒，包括非斑块形成病毒。随后，该方案可用于ZIKV感染的研究，并可用于检测其他临床上重要的病毒。

ZIKV可以使用FFA进行量化，如图3所示。对于 24 孔板，将感染的 Vero 细胞固定在感染后 48 小时、60 小时、72 小时、84 小时和 96 小时。结果显示，感染后96小时（4天）后，细胞保持完整（未观察到细胞脱离）（图4和补充图8A-E）。在感染后48小时（2天）首次观察到病毒病灶的出现（图4A-F）。然而，病灶尺寸?...

Watch this Scientific Journal Video about Virus Propagation and Cell-Based Colorimetric Quantification at JoVE.com

Virus Propagation and Cell-Based Colorimetric Quantification

本方案描述了寨卡病毒（ZIKV）在Vero非洲绿猴肾细胞中的繁殖，以及使用基于细胞的比色免疫检测方法以24孔和96孔（高通量）形式对ZIKV进行定量。

病毒传播和基于细胞的比色定量

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

该协议能够在不依赖细胞病变效应的情况下识别和定量寨卡病毒。这取决于病毒成分的抗体识别。使用病毒特异性抗体有助于识别混合人群中的不同病毒血清型。该方法比经典的斑块形成测定法具有优势。当与自动成像系统一起应用以实现快速细胞计数时，它速度更快，并且能够支持高通量应用。所提出的协议具有很强的适应性。通过适当的修改，所提出的方案可以应用于各种细胞类型和病毒靶标。首先在 75 平方=厘米的细胞培养瓶中培养 Vero 细胞，该培养瓶含有 12 毫升补充有 10% FBS 和 2 毫摩尔 L-谷氨酰胺的 DMEM。将烧瓶放入37摄氏度和5%二氧化碳的细胞培养箱中。要感染细胞单层，请使用 10 毫升血清移液管从细胞培养瓶中除去生长培养基。使用五毫升血清移液管，用三毫升DPBS冲洗烧瓶两次。接下来，将 2 毫升无血清 DMEM 和 20 微升寨卡病毒接种物加入细胞培养瓶中。让烧瓶在室温下孵育一小时，轻轻摇晃以促进病毒吸附。在孵育结束时，使用5毫升血清移液管，小心地从细胞培养瓶中取出并丢弃稀释的病毒接种物。用三毫升DPBS冲洗细胞培养瓶两次。然后，将12毫升维持培养基加入细胞培养瓶中以维持感染的细胞。将受感染的Vero细胞在37摄氏度和5%二氧化碳的细胞培养箱中孵育三天。孵育三天后，使用10毫升血清移液管将含有寨卡病毒的细胞培养上清液收集到50毫升离心管中。对于病毒定量，将 Vero 细胞接种在指定的培养皿中，并让它们在 37 摄氏度和 5% 二氧化碳气氛下生长过夜。为每个板准备六个无菌 1.5 毫升微量离心管以执行 10 倍的连续稀释，包括用于阴性对照的额外管。对于 24 孔板设置，向所有六个微量离心管中加入 450 μL 无血清 DMEM。对于 96 孔板设置，将 135 μL 无血清 DMEM 分配到另外六个试管中。为了对 24 孔板实验进行连续稀释，将 50 微升寨卡病毒原液加入 10 至减去一个含有 450 微升无血清 DMEM 的试管中。对于 96 孔板实验，将 15 微升寨卡病毒原液加入 10 至减去一个含有 135 微升无血清 DMEM 的试管。涡旋每个试管以彻底混合病毒和培养基，确保病毒颗粒在稀释液内均匀分布。使用新鲜的移液器吸头，将10至减一管重悬，并将50和15微升稀释的寨卡病毒转移到10至负两管中，分别作为24孔板和96孔板的第二个十倍稀释液。取出并丢弃相应板的每个孔的条件培养基。用DPBS冲洗每个孔两次以去除任何残留物。从最高稀释度开始，将连续稀释的病毒接种物加入孔中，努力实现最低稀释度。孵育一小时后，从孔中取出并丢弃病毒悬浮液，从最低浓度到最高浓度开始。用DPBS洗涤受感染的细胞两次，以去除任何病毒悬浮的痕迹。用DMEM和1.5%低粘度羧甲基纤维素覆盖孔。将板在37摄氏度的细胞培养箱中用5%二氧化碳孵育。孵育后，取出并弃去覆盖培养基，并用PBS洗涤细胞三次。对于 96 孔板，使用多通道移液器去除并弃去覆盖介质，并用每孔 60 μL PBS 洗涤细胞 3 次。加入4%多聚甲醛固定细胞，并将板在室温下孵育20分钟。20分钟后，弃去多聚甲醛并用PBS洗涤细胞三次。接下来，加入3,3'二氨基联苯胺过氧化物酶底物，并在黑暗中孵育板30分钟。30分钟后，用水清洗井停止反应。将板风干过夜，然后进行焦点计数。计算所选稀释液的每个重复的病灶，并计算每个病灶的平均数。感染后的Vero细胞在感染后的不同时间点被固定。对于 2 孔板，在感染后 48 小时首次观察到病毒病灶的出现，尽管病灶大小太小而无法准确计数。感染后96小时后，没有细胞脱离的迹象。在感染后 60 小时，病灶大小已增加到计数的最佳水平。随后，随着时间的流逝，病灶变大，强度开始相互合并或重叠，形成随时间增长的簇。因此，选择感染后 60 小时形成的病灶来测定 24 孔板中的寨卡病毒滴度。对于 96 孔板，细胞在感染后 72 小时后保持完整。病毒病灶的出现是在感染后 24 小时首次观察到的。然而，在感染后长达 36 小时，病灶大小太小。在感染后 48 小时达到最佳病灶大小。在后一个时间点，观察到重叠或合并的病灶，并且重叠的病灶数量随时间增加。选择感染后 48 小时形成的病灶以确定寨卡病毒分离株的病毒滴度。在每个孔的侧面轻轻添加 DPBS，并前后摇晃板一到三次，以去除细胞碎片和多余的培养基。这项技术将极大地有利于寨卡病毒研究的研究人员，并且还可以广泛地用于量化其他临床上重要的病毒，使其成为病毒监测和诊断的宝贵工具。

Research

JoVE Journal

Immunology and Infection

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

科研

免疫与感染

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

传播<em&gt; Homalodisca coagulata病毒01</em&gt;通过<em&gt; Homalodisca vitripennis</em&gt;细胞培养

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

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 ...

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

猪全血表达编码和非编码 rna 类的鉴定

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

体外ELISA测试评估狂犬病疫苗效力

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...