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Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the development of disease. This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. As VZV is highly cell-associated in tissue culture, the reporter virus inoculum described here is a preparation of infected cells. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication.

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VZV Replication Assays
水痘带状疱疹复制分析

微生物学 > 微生物-宿主相互作用 > 体外实验模型 > 细胞系
作者: Samantha J. Griffiths
Samantha J. GriffithsAffiliation: Division of Pathway and Infection Medicine, University of Edinburgh, Edinburgh, UK
For correspondence: samantha.griffiths@ed.ac.uk
Bio-protocol author page: a1579
 and Jürgen Haas
Jürgen HaasAffiliation: Division of Pathway and Infection Medicine, University of Edinburgh, Edinburgh, UK
Bio-protocol author page: a1580
Vol 4, Iss 16, 8/20/2014, 3297 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1210

[Abstract] Varicella zoster virus (VZV) is a human herpesvirus which causes Varicella (chickenpox) upon primary infection and Zoster (shingles) following reactivation from latency (von Bokay, 1909). Whilst VZV is extensively studied, inherent features of VZV replication, such as cell-association of virus particles during in vitro culture and a restricted host range (limited to humans and some other primates) mean the cellular and viral mechanisms underlying VZV reactivation and pathogenesis remain largely uncharacterised. Much remains to be learnt about VZV, interactions with its host, and the development of disease. This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. As VZV is highly cell-associated in tissue culture, the reporter virus inoculum described here is a preparation of infected cells. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication.

[Abstract]

Materials and Reagents

  1. VZV-permissive human cells (e.g. MeWo cells) (ATCC, catalog number: HTB-65 )
  2. Minimal essential medium eagle with Earle’s BBS, with L-glutamine (Lonza, catalog number: 12-611F )
  3. Fetal bovine serum (FBS) (LabTech, catalog number: FCS-SA-10454 )
  4. Penicillin: streptomycin (5,000 units/ml each) (Lonza, catalog number: DE17-603E )
  5. Non-essential amino acids (NEAA) (Life Technologies, catalog number: 11140035 )
  6. 1x Trypsin-EDTA liquid (0.05% Trypsin, 0.53 mM EDTA-4Na) (Life Technologies, catalog number: 25300096 )
  7. Phosphate buffered saline without Magnesium or Calcium (Lonza, catalog number: 17-516F )
  8. Recombinant VZV-GFP cell-associated virus stock (Zerboni, 2000)
  9. MeWo growth medium (see Recipes)

Equipment

  1. 75 cm2 filter cap tissue culture flasks (Sigma-Aldrich, catalog number: C7106-120EA )
  2. 96-well black tissue culture-treated plates (48/case) (VWR International, catalog number: 734-1609 )
  3. 50 ml centrifuge tube (e.g. Corning, catalog number: 430290 )
  4. Disposable hemocytometer (KOVA Glasstic slide 10 with counting grid) (HYCOR Biomedical, catalog number: 87144E )
  5. Fluorescent plate reader (e.g. POLARstar OPTIMA, BMG LABTECH)
  6. Class II Microbiological safety cabinet
  7. Humidified cell culture incubator (37 °C, 5% CO2)
  8. Pipette aid (Alpha laboratories, catalog number: 4-131-201-E )
  9. Centrifuge (e.g. Eppendorf, catalog number: 5811-000.010 )
  10. Light microscope

Procedure

  1. Take a semi-confluent (~50%) T75 flask of MeWo cells that has been split the previous day (see Note 1).
  2. Remove media, rinse gently in sterile phosphate buffered saline (PBS), and discard wash.
  3. Dislodge cells by adding 3 ml trypsin. Incubate for around 5 min at 37 °C or until cells are fully dislodged.
  4. Inactivate trypsin by adding 7 ml MeWo growth medium.
  5. Pipette up and down to create a single-cell suspension.
  6. Remove 12 μl and count cell density in a disposable hemocytometer.
  7. Dilute cells to a density of 2 x 105 cells/ml in MeWo growth medium and seed 100 μl per well in a black, flat-bottomed 96-well plate to give 2 x 104 cells/well (see Note 2).
  8. Leave cells to adhere overnight in a humidified incubator with 5% CO2 at 37 °C.
  9. Remove a vial of VZV-GFP of known titre from liquid nitrogen storage and thaw immediately in a water bath at 37 °C until just thawed (see Note 3).
  10. Transfer thawed cells to a 50 ml centrifuge tube and slowly add 30 ml growth medium (pre-warmed to 37 °C) drop by drop to gradually increase the temperature of the cells.
  11. Centrifuge for 10 min at 200 x g to pellet cells.
  12. Gently remove the entire washing medium and resuspend in 1 ml growth media.
  13. Dilute the cells in sufficient growth media to result in 1,000 IU/ml (see Note 4).
  14. Remove plates from the incubator, and remove media by inverting plate and shaking over a container of suitable disinfectant.
  15. Add 100 μl of the washed and resuspended VZV-GFP cell inoculum to each well to be infected, and 100 μl growth medium to uninfected control wells (see Note 5).
  16. Replace the lid on the plate and return the plate back to the incubator. Monitor virus replication as a measure of GFP fluorescence from ~22 h post-infection until replication plateaus (see Note 6).
  17. For comparison of virus replication in untreated and treated cells (protein overexpression, gene depletion, drug treatment etc.) calculate the replication slope over the linear growth phase and normalise treated cells to untreated (“normal” replication; Figure 1).


    Figure 1. Calculation of replication slopes. A. Virus growth is monitored over multiple rounds of replication as a function of GFP fluorescence. B. Relative fluorescence units for mock transfected and positive controls-transfected (IDE, a cellular receptor for VZV; ORF28, the DNA polymerase for VZV) cells is plotted against hours post-infection to generate growth curve. C. The slope of replication over the linear phase of growth is calculated in Excel using the linest equation, and normalized to control (mock) transfected cells. D. Replication slopes are compared in a bar chart to see the effect of siRNA gene depletion on VZV replication.

Notes

  1. Virus replication proceeds more efficiently in cells that have been freshly passaged before seeding into assay plates.
  2. Using a black assay plate will help reduce background and crosstalk between wells.
  3. When grown in culture VZV virus particles remain associated with the cell membrane. As such, VZV-infected MeWo cells are used as the inoculum for VZV replication assays. These cells are stored in liquid nitrogen and need to be thawed and washed before use. Cells are extremely sensitive to temperature changes. When thawing it is essential that they are thawed quickly at 37 °C until only just thawed (1-2 min). Do not leave cells for longer periods of time as this will considerably reduce cell viability and therefore titre of replication-competent virus.
  4. It is important to do the infection with a known quantity of virus (multiplicity of infection; MOI) to generate growth curve over a reasonable time-scale. The titre [number of infectious units per ml of virus (IU/ml)] of VZV-GFP-infected MeWo cells is determined by a standard plaque assay, where a virus stock is added to a Mewo cell monolayer and overlaid with agarose. This results in the infection only of adjacent cells, which subsequently die to leave an empty patch within the cell monolayer. These patches, or ‘plaques’ can be counted to quantify the virus as ‘plaque-forming units’, or infectious units (IU), per ml inoculum. In our experience 100 IU per well of a 96-well plate produces a suitable growth curve for replication analyses and comparisons. For example, a virus stock with a titre of 6.6 x 104 IU/ml would contain 6,600 IU in 100 µl. A 1 ml aliquot of virus inoculum should therefore be resuspended in 66 ml media.
  5. Tissue culture plasticware, cells and growth medium all have some level of fluorescence. When utilising fluorescent reporter genes it is therefore essential to have appropriate controls to provide background fluorescence readings. For this assay, the fluorescence from mock-infected cells is used as background.
  6. Replication is monitored over regular intervals to enable a complete growth curve (fluorescence over time) to be plotted. When comparing VZV replication between untreated and treated samples we use the slope of replication over the linear growth phase. It is therefore important to have as many measurements over this phase as possible (with a minimum of 6 for statistical reliability of the slope calculation). A test replication assay with your own equipment will allow the time of linear growth to be established, and assay timings can be adjusted to ensure measurements can be taken over this period.

Recipes

  1. MeWo growth medium
    Eagle’s minimum essential medium (EMEM)
    5% FCS
    1% p-s
    1% NEAA

Acknowledgments

We are grateful to Prof. Ann Arvin (Stanford School of Medicine) for the kind gift of the VZV-GFP clone, and to Dr. Armin Baiker for the propagation of a cell-associated VZV-GFP virus stock. The protocol was optimised by Dr Lakshmi N. Kaza and Dr Samantha Griffiths. The authors gratefully acknowledge the MRC for funding (G0501453 J.H.).

References

  1. von Bokay, J. (1909). Uëber den aëtiologischen Zusammenhang der Varizellen mit gewissen Fällen von Herpes Zoster. Wien Klin Wochenschr 22: 1323-1326.
  2. Zerboni, L., Sommer, M., Ware, C. F. and Arvin, A. M. (2000). Varicella-zoster virus infection of a human CD4-positive T-cell line. Virology 270(2): 278-285.

材料和试剂

  1. VZV允许的人细胞(例如MeWo细胞)(ATCC,目录号:HTB-65)
  2. 含有Earle's BBS,含L-谷氨酰胺的最小必需培养基(Lonza,目录号:12-611F)
  3. 胎牛血清(FBS)(LabTech,目录号:FCS-SA-10454)
  4. 青霉素:链霉素(每种5,000单位/ml)(Lonza,目录号:DE17-603E)
  5. 非必需氨基酸(NEAA)(Life Technologies,目录号:11140035)
  6. 1x胰蛋白酶-EDTA液体(0.05%胰蛋白酶,0.53mM EDTA-4Na)(Life Technologies,目录号:25300096)
  7. 不含镁或钙的磷酸盐缓冲盐水(Lonza,目录号:17-516F)
  8. 重组VZV-GFP细胞相关病毒原种(Zerboni,2000)
  9. MeWo生长培养基(参见食谱)

设备

  1. 75cm 2过滤盖组织培养烧瓶(Sigma-Aldrich,目录号:C7106-120EA)中。
  2. 96孔黑色组织培养处理板(48 /箱)(VWR International,目录号:734-1609)
  3. 50ml离心管(例如,Corning,目录号:430290)
  4. 一次性血细胞计数器(KOVA Glasstic slide 10 with counting grid)(HYCOR Biomedical,目录号:87144E)
  5. 荧光板读数器(例如 POLARstar OPTIMA,BMG LABTECH)
  6. II类微生物安全柜
  7. 加湿的细胞培养孵育器(37℃,5%CO 2)
  8. 移液器辅助(Alpha实验室,目录号:4-131-201-E)
  9. 离心机(例如:Eppendorf,目录号:5811-000.010)
  10. 光学显微镜

程序

  1. 取半天汇合(约50%)T75烧瓶的MeWo细胞,前一天已分裂(见注1)。
  2. 取出培养基,用无菌磷酸盐缓冲液(PBS)轻轻冲洗,弃去洗液
  3. 通过加入3毫升胰蛋白酶排除细胞。 在37℃孵育约5分钟或直到细胞完全移动
  4. 通过加入7ml MeWo生长培养基来灭活胰蛋白酶
  5. 移液器上下移动以产生单细胞悬浮液。
  6. 去除12微升,并在一次性血细胞计数器中计数细胞密度
  7. 在MeWo生长培养基中将细胞稀释至2×10 5个细胞/ml的密度,在黑色平底96孔板中每孔加入100μl种子,得到2×10 4个 细胞/孔(见注2)
  8. 使细胞在37℃下在具有5%CO 2的潮湿培养箱中粘附过夜。
  9. 从液氮存储中取出一瓶已知滴度的VZV-GFP,立即在37℃的水浴中解冻,直到解冻(见注3)。
  10. 将解冻的细胞转移到50ml离心管中,并逐滴缓慢加入30ml生长培养基(预热至37℃)逐渐增加细胞的温度。
  11. 在200×g离心10分钟以沉淀细胞
  12. 轻轻取出整个洗涤介质,并重悬于1ml生长培养基中
  13. 在足够的生长培养基中稀释细胞,产生1000 IU/ml(见注4)
  14. 从培养箱中取出培养板,通过倒置培养板并在合适的消毒剂容器上摇动来除去培养基
  15. 向每个待感染的孔中加入100μl经洗涤和重悬的VZV-GFP细胞接种物,并向未感染的对照孔中加入100μl生长培养基(参见注释5)。
  16. 更换板上的盖子,并将板放回培养箱。监测病毒复制,作为感染后〜22小时至复制平台期间GFP荧光的量度(见注6)。
  17. 为了比较未处理和处理的细胞中的病毒复制(蛋白质过表达,基因耗竭,药物处理等),计算在线性生长期的复制倾向,并将处理的细胞标准化为未处理的("正常"复制;图1)。


    图1.复制斜率的计算。A.作为GFP荧光的函数,在多轮复制中监测病毒生长。 B.将感染后模拟转染和阳性对照(IDE,VZV的细胞受体; ORF28,VZV的DNA聚合酶)细胞的相对荧光单位相对于感染后的小时绘图以产生生长曲线。 C.在Excel中使用线性方程计算复制对生长的线性阶段的斜率,并相对于对照(模拟)转染的细胞标准化。 D.在条形图中比较复制斜率,以观察siRNA基因耗尽对VZV复制的影响。

笔记

  1. 病毒复制在接种到测定板之前新鲜传代的细胞中更有效地进行
  2. 使用黑色测定板有助于减少孔之间的背景和串扰
  3. 当在培养物中生长时,VZV病毒颗粒保持与细胞膜结合。因此,VZV感染的MeWo细胞用作VZV复制测定的接种物。这些细胞储存在液氮中,需要在使用前解冻和洗涤。细胞对温度变化非常敏感。当解冻时,它们必须在37℃下快速解冻,直到仅仅解冻(1-2分钟)。不要将细胞留在更长的时间,因为这将大大降低细胞活力,因此降低复制能力的病毒的滴度。
  4. 用已知量的病毒(感染复数; MOI)进行感染以在合理的时间尺度上产生生长曲线是重要的。通过标准噬菌斑测定法测定VZV-GFP感染的MeWo细胞的滴度[每ml病毒的感染单位数(IU/ml)],其中将病毒原种加入Mewo细胞单层并用琼脂糖覆盖。这导致仅感染相邻细胞,其随后死亡以在细胞单层内留下空补片。可以计数这些斑块或"噬菌斑"以将病毒定量为每ml接种物的"噬斑形成单位"或感染单位(IU)。根据我们的经验,96孔板的每孔100IU产生用于复制分析和比较的合适的生长曲线。例如,滴度为6.6×10 4 U/IU/ml的病毒原液将在100μl中含有6,600IU。因此,1ml等分的病毒接种物应重悬于66ml培养基中
  5. 组织培养塑料制品,细胞和生长培养基都具有一定程度的荧光。当使用荧光报告基因时,因此必须具有适当的对照以提供背景荧光读数。对于该测定,将来自模拟感染细胞的荧光用作背景
  6. 在规则的时间间隔上监测复制以使得能够绘制完整的生长曲线(随时间的荧光)。当比较未处理和处理的样品之间的VZV复制时,我们使用在线性生长期的复制的斜率。因此,重要的是在该相位上尽可能多地进行测量(对于斜率计算的统计可靠性,具有最小值6)。使用您自己的设备进行的测试复制测定将允许建立线性生长的时间,并且可以调整测定时间以确保在这段时间内进行测量。

食谱

  1. MeWo生长培养基
    鹰的最低必需培养基(EMEM)
    5%FCS
    1%p-s
    1%NEAA

致谢

我们感谢安阿文教授(斯坦福医学院)的VZV-GFP克隆的恩赐,并感谢博士Armin Baiker传播细胞相关的VZV-GFP病毒股票。 该方案由Lakshmi N. Kaza博士和Samantha Griffiths博士进行了优化。 作者衷心感谢MRC的资助(G0501453 J.H.)。

参考文献

  1. von Bokay,J。(1909)。 UëberdenaëtiologischenZusammenhang der Varizellen mit gewissenFällenvon Herpes Zoster。 Wien Klin Wochenschr 22:1323-1326。
  2. Zerboni,L.,Sommer,M.,Ware,C.F.and Arvin,A.M。(2000)。 人类CD4阳性T细胞系的水痘带状疱疹病毒感染。 em> Virology 270(2):278-285
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How to cite this protocol: Griffiths, S. J. and Haas, J. (2014). VZV Replication Assays. Bio-protocol 4(16): e1210. DOI: 10.21769/BioProtoc.1210; Full Text



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