Name: plaquing of herpes simplex viruses
Uploaded: 2021-11-05T13:00:00
Description: Watch this Scientific Journal Video about Plaquing of Herpes Simplex Viruses at JoVE.com

我们感谢我们实验室（PJD和BJM）的无数学生，他们多年来一直与我们一起改进这些方法。特别感谢Stan Person，在他的指导下，这种方法首先得到了发展。这项工作得到了陶森大学费舍尔科学与数学学院本科生研究支持基金和NIGMS Bridges的部分支持，获得了学士学位补助金5R25GM058264。此内容完全是作者的责任，并不一定代表美国国立卫生研究院国家普通医学科学研究所的官方观点。

Vero细胞和活疱疹病毒的所有程序均已获得陶森大学机构生物安全委员会的批准。这些过程的一般化方案如图 1 所示。
1. Vero细胞的接种
<ol><li>在开始斑块测定的前一天，胰蛋白酶消化Vero细胞，并按照标准细胞培养方法将其重悬于常规Dulbecco的改良鹰培养基（DMEM）和补充剂中19。将胰蛋白酶消化的细胞重悬于10mL DMEM中，每?...

<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=Production+of+malignancy+in+vitro;+IV.+The+mouse+fibroblast+cultures+and+changes+seen+in+the+living+cells.">Production of malignancy in vitro; IV. The mouse fibroblast cultures and changes seen in the living cells.</a> Journal of the National Cancer Institute. 4 (2), 165-212 (1943).</li><li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+problems+of+animal+virology+as+studied+by+the+plaque+technique.">Some problems of animal virology as studied by the plaque technique.</a> Cold Spring Harbor Symposia on Quantitative Biology. 18, 273-279 (1953).</li><li>Enders, J. F., Weller, T. H., Robbins, F. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+viruses+types+1+and+2.+Type+and+strain+specific+characteristics+affecting+virus+plaque+formation.">Herpes simplex viruses types 1 and 2. Type and strain specific characteristics affecting virus plaque formation.</a> Arch Gesamte Virusforsch. 46 (1-2), 36-43 (1974).</li><li>Muratore, O., Tonoli, E. 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P., Eliceiri, G. L., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+ribosome+in+mouse-hamster+hybrid+cells.">Two types of ribosome in mouse-hamster hybrid cells.</a> Nature New Biology. 230 (10), 52-54 (1971).</li><li>Giannasca, N. J., Suon, J. S., Evans, A. C., Margulies, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-based+controlled+release+delivery+of+acyclovir+from+poly-(ethylene+co-vinyl+acetate)+rings.">Matrix-based controlled release delivery of acyclovir from poly-(ethylene co-vinyl acetate) rings.</a> Journal of Drug Delivery Science and Technology. 55, 101391 (2020).</li><li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. 7th edn. , (2016).</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 Science, USA. 38 (8), 747-752 (1952).</li><li>Baer, A., Kehn-Hall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+concentration+determination+through+plaque+assays:+using+traditional+and+novel+overlay+systems.">Viral concentration determination through plaque assays: using traditional and novel overlay systems.</a> Journal of Visualized Experiments. (93), e52065 (2014).</li><li>Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+low-viscosity+overlay+medium+for+viral+plaque+assays.">New low-viscosity overlay medium for viral plaque assays.</a> Virology Journal. 3, 63 (2006).</li><li>Culley, S., Towers, G. J., Selwood, D. L., Henriques, R., Grove, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+counter:+Automated+quantification+of+in+vitro+virus+replication+by+fluorescence+microscopy.">Infection counter: Automated quantification of in vitro virus replication by fluorescence microscopy.</a> Viruses. 8 (7), 201 (2016).</li><li>Hudu, S. A., 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=Quantitative+Hepatitis+B+e+antigen:+A+better+predictor+of+Hepatitis+B+virus+DNA+than+quantitative+Hepatitis+B+surface+antigen.">Quantitative Hepatitis B e antigen: A better predictor of Hepatitis B virus DNA than quantitative Hepatitis B surface antigen.</a> Clinical Laboratory. 64 (4), 443-449 (2018).</li><li>Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-dependent+DNA+polymerase+in+virions+of+RNA+tumour+viruses.">RNA-dependent DNA polymerase in virions of RNA tumour viruses.</a> Nature. 226 (5252), 1209-1211 (1970).</li><li>Scolnick, E. M., Aaronson, S. A., Todaro, G. J., Parks, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+dependent+DNA+polymerase+activity+in+mammalian+cells.">RNA dependent DNA polymerase activity in mammalian cells.</a> Nature. 229 (5283), 318-321 (1971).</li><li>Strick, L. B., Wald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+herpes+simplex+virus:+is+PCR+the+new+gold+standard.">Diagnostics for herpes simplex virus: is PCR the new gold standard.</a> Molecular Diagnosis and Therapy. 10 (1), 17-28 (2006).</li><li>Ramakrishnan, M. 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虽然斑块测定几乎与哺乳动物细胞培养物本身一样古老，但似乎每个实验室都有自己的一套方案来执行这种基本测定5，6，10，11，12，13，14，15，16，20...

病毒斑块测定的开始可以追溯到 19 世纪 90 年代首次发现病毒1。首先分离出烟草花叶病毒并传递到烟叶上，其中可以识别和量化单个感染点，其起源于单个活病毒实体2，后来被鉴定为病毒粒子2。后来对细菌和噬菌体的开创性研究完善了用于斑块这些病毒的技术，包括生长中期的细菌，噬菌体样品的连续稀释，以及随后可视化细菌草坪中的字面孔（命名为斑块）的顶部琼脂3。
动物病毒的攀爬滞了对噬菌体进行的激动人心的研究，主要是因为培养哺乳动物细胞所需的方法直到20世纪40年代才得到开发4。然而，在没有整个宿主生物体的情况下，生长中的鼠细胞的出现4催生了培养和计数病毒能力的新时代。这项工作在鸡细胞中传播和定量西马脑脊髓炎病毒和在人细胞中脊髓灰质炎病毒5，6。随着可培养哺乳动物细胞领域的扩大，不同宿主细胞对各种病毒感染的依赖为世界提供了研究各种病毒的可能性7。这包括人类疱疹病毒的传播和定量，特别是单纯疱疹病毒-1（HSV-1）和-2（HSV-2），它们会导致皮肤黏膜病变8。重要的是，所有斑块测定都依赖于活病毒体的存在，其可以在样品中以受体介导的方式进入宿主细胞9。尽管关于执行斑块测定的出版物无处不在和众多5，10，11，12，13，14，15，16，这些用于HSV-1 / -2的方法都是艺术和科学的混合体;如果不适当注意方案中的每个细节，就无法进行测定，也不能在没有对过程中的微妙之处进行严格观察的情况下执行成功的测定。该手稿描述了HSV-1/-2斑块测定最一致的可重复性方法之一，并具有很少讨论的测定技术的精确细节。
目前的方案可靠地获得HSV-1和-2的活斑块形成单元（PFU）计数。使用低传代（低于传代号155）的Vero细胞（转化的非洲绿猴肾上皮细胞）获得最佳结果，并在补充了10 %胎儿小牛血清（FCS），L-丙氨酰-L-谷氨酰胺和抗生素/抗真菌混合物的α-MEM17中常规生长18。Vero细胞标准地在这种培养基中繁殖，每周两到三次，每次 稀释1/5 。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-MEM</td>
			<td>Lonza</td>
			<td>12169F</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystal violet</td>
			<td>Alfa Aesar</td>
			<td>B2193214</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (no Mg++ or Ca++)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Millipore-Sigma</td>
			<td>TMS-013-B</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine (Glutamax)</td>
			<td>Gibco</td>
			<td>GS07F161BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Thermo Fisher</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Millipore-Sigma</td>
			<td>27-441-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Quaternary agent (Lysol I.C.)</td>
			<td>Thermo Fisher</td>
			<td>NC9645698</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Corning</td>
			<td>25900CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vero cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td></td>
		</tr>
	</tbody>
</table>

plaquing of herpes simplex viruses

有许多已发表的用于诱捕病毒的方案，包括主要文献中关于方法学的参考文献。然而，Plaquing病毒可能很难执行，需要专注于其规格和改进。对于新生来说，掌握这是一种非常具有挑战性的方法，主要是因为它需要对最微小的细节进行细致的关注。这种诱发单纯疱疹病毒的演示应该可以帮助那些多年来一直在努力可视化该方法的人，尤其是其细微差别。虽然本手稿基于标准绦塑方法的相同原则，但它的不同之处在于它包含以下详细说明：（1）如何最好地处理宿主细胞以避免在此过程中的中断，（2）比琼脂糖更有用的粘性培养基来限制病毒粒子的扩散，以及（3）简单的固定和染色程序，产生可靠的可重复结果。此外，随附的视频有助于展示过程中更精细的区别，在指导他人进行斑块测定时经常会错过这些区别。

表1显示了具有最佳结果的实验。所有10倍稀释后，斑块计数减少约10倍。这些类型的数据也可以在图2中看到，这是一种实际的斑块测定，其中所有三个重复的可计数斑块数量都在10-4范围内。在图3的顶行中可以看到相同的情况，其中可计数的斑块数在10-3稀释液中。
然而， 底行图3...

Watch this Scientific Journal Video about Plaquing of Herpes Simplex Viruses at JoVE.com

Plaquing of Herpes Simplex Viruses

Plaquing是一种用于量化人群中活病毒的常规方法。虽然在各种微生物学课程中经常教授细菌和噬菌体的诱捕，但哺乳动物病毒的幽射更为复杂和耗时。该协议描述了可靠地用于单纯疱疹病毒常规工作的程序。

单纯疱疹病毒的扁平化

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

该协议可帮助学生清楚地识别个体感染并轻松进行测定，从而更有效地量化病毒滴度。该协议结合了简单的固定和染色程序，可产生可靠且可重复的结果。这种方法可以深入了解抗病毒治疗疾病的准确性和有效性。这种整理方案更困难的障碍是接种板的细胞计数，确保细胞在整个过程中不会过度拥挤，干燥或被划伤。人们也可能发现该协议的整体细致性质有困难。然而，实践是这个实验成功的主要贡献者。首先，在一次实验室会话中将病毒添加到细胞中完成样品稀释。连续稀释在PBS中斑块的每个病毒样品。通常使用 10 倍稀释液进行最精确的跟踪。将所有病毒稀释液保持在冰上，直到准备好将这些稀释的病毒样品添加到细胞中。使用移液器从一个或两个孔中取出细胞培养基。小心地将稀释的病毒样品滴加到每个单层细胞中，通过每个孔的侧面滴加。每一个或两个孔重复该过程，直到整个板中充满被斑块的病毒样品。用手轻轻摇动整个盘子。然后将板在37摄氏度下孵育一小时，以使病毒吸附。每10分钟后，从培养箱中取出培养皿，再次轻轻摇晃以将病毒更均匀地传播到每个孔中，然后将其放回培养箱中。为了减少无意中计数未吸收的接种物的可能性，请使用1000微升移液器吸头从孔中取出病毒样品，并将样品丢弃在废烧杯中。将2.5毫升甲基纤维素覆盖物放在板上，然后将其放回培养箱中。对废烧杯进行消毒，通常添加漂白剂，碘伏或类似的杀病毒剂，然后将其从生物安全柜中取出。在为期两天的孵育后，使用移液器一次从一个或两个孔中取出甲基纤维素覆盖物，并将其放入废烧杯中。向每个孔中加入约2毫升1%结晶紫在50%乙醇中。将板中装满污渍的所有孔在37摄氏度下孵育30分钟。此时，如前所述，对废烧杯进行消毒。用自来水大力清洗板，直到径流清除。确保每个稀释系列的斑块数量差异约为10倍。该图描绘了斑块计数减少10倍，其中所有三个重复的可计数斑块数量都在10到负4范围内。图中的前三幅图像描绘了斑块在10到负3稀释。然而，当斑块测定未良好进行时，底行显示结果。从顶部周围可见区域的任何稀释中可以看到很少的斑块，Vero细胞已完全从底物中抬起。同样，该图显示了斑块测定过程中细胞干燥或处理不当的问题。然而，这个数字严重表明Vero细胞接种不良。每个孔都显示出较深的紫色斑点，表明细胞没有很好地分布在整个孔中，并且成簇堆叠在一起。为获得最佳结果，请确保病毒均匀地分布在板上，以避免斑块聚集斑块测定比其他定量方法更有效，因为它只计算活病毒。这样，就可以建立真正的抗病毒功效。此外，这项技术使我们能够量化污染物上的病毒存在，并探索我们疱疹病毒研究领域内新型医疗设备药物递送的有效性。

Research

JoVE Journal

Immunology and Infection

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

科研

免疫与感染

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...