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Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses
α疱疹病毒的疫源地分析和感染复数(MOI)计算   

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Abstract

Titration of viral stocks is a critical process before any experimental use of the virus. Here we describe an infectious focus assay for several alphaherpesviruses, a titration method for fluorescently labeled viruses, based on the original plaque assay. In addition, the calculation of multiplicity of infection (MOI) is presented.

Keywords: Multiplicity of infection(多重感染), Viral infection(病毒感染), Herpesvirus(疱疹病毒)

Materials and Reagents

  1. Standard cell line [e.g. MeWo (ATCC® HTB-65 TM); ARPE19 (ATCC® CRL-2302TM); Vero (ATCC® CCL-81 TM)]
  2. Fluorescently labeled virus stock (genetically engineered VZV-66GFP, HSV1-gC-GFP and PRV-IE180-GFP were kindly provided by Prof. Paul R. Kinchington, University of Pittsburgh, Pittsburgh PA)
  3. Growth medium for the cells listed above (e.g. item #6, Fibroblast medium)
    Note: However, it can be any other medium for other pairs of virus and cells.
  4. 4% paraformaldehyde (EMS 15710) in phosphate buffered saline with divalent cations (e.g. Biological Industries, catalog number: 020201A )
  5. High glucose DMEM (Biological Industries, catalog number: 010551A )
  6. Fetal calf serum (Biological Industries, catalog number: 041271A )
  7. Glutamine (Biological Industries, catalog number: 030201 )
  8. Penicillin-streptomycin (Biological Industries, catalog number: 030311 )
  9. Agarose low EEO (Hispanagar, catalog number: D1500 )
  10. Fibroblast medium (see Recipes)
  11. Overlaying media for free virus (see Recipes)
  12. Crystal violet (Sigma-Aldrich, catalog number: C0775 ) (see Recipes)

Equipment

  1. 24-well tissue culture plates
  2. Incubator 37 °C, 5% CO2
  3. Epifluorescent inverted microscope

Procedure

  1. Cell-associated varicella zoster virus (VZV)
    1. Grow MeWo or ARPE19 cells in a 24 well plate until they reach 90% confluence (~300,000 or 200,000 cells/well when seeding ~24 h before the infection, respectively). Use 500 µl/well volume to grow and infect the cells. The use of MeWo cells for VZV is documented in Heineman and Cohen (1994), Eisfeld et al. (2007) and Markus et al. (2011).
    2. Prepare 10-fold serial dilutions of VZV-infected MeWo cells. Typically, the range of the dilutions is 10-1 to 10-6 of the initial stock. The volume inoculated to each well can range up to 500 µl.
    3. Add the infected cells to the 24-well plate containing the 90% confluent naïve cell cultures in triplicate and incubate at 37 °C for at least 2 h. There is no special need to replace the medium after the addition of the inoculum, therefore the infected-cells can be left overnight as well. No agar overlay is required as no secondary plaques are formed by VZV, which does not release virions into the overlaying medium.
    4. Using a 10x objective with a fluorescent microscope, count all the fluorescent foci (groups of fluorescent cells) in each well after 2-5 days. Take care not to count the same fluorescent focus twice, and screen all the wells in the same order. This process can be automated with a microscope equipped with a computerized stage and image analysis software. If all fluorescent foci have developed into plaques then units used for the final virus concentration are plaque forming units (PFU).
    5. To calculate the PFU/ml, use the average number of foci in the wells with an input virus dilution resulting in 10-100 foci. For example, if the 10-3 dilution resulted in 24, 26 and 30 fluorescent foci per well, and the inoculated amount was 100 µl, then the stock concentration is


  2. Cell-free VZV
    1. Grow ARPE19 cells in a 24 well plate until they reach 90% confluence (~200,000 cells/well when seeding ~24 h before the infection). These cells are more easily infected at low VZV concentrations as compared to MeWo or MRC5 cells (Schmidt-Chanasit et al., 2008; Pasdeloup et al., 2013). Use 500 µl/well volume to grow and infect the cells.
    2. Prepare 5-fold serial dilutions of cell-free VZV in high glucose DMEM. In our experience, diluting the virus in growth medium for cells which are not sensitive to the presence of serum does not seem to affect viral infectivity. Typically, the range of the dilutions is 1:5 to 1:625 of the initial stock.
    3. Inoculate the ARPE19 cells with 100-150 µl/well of the viral dilutions in triplicates.
    4. In a CO2 incubator, let the virus adsorb to the cell monolayer for 2 h, agitating occasionally.
    5. Remove the inoculum and add 0.5 ml pre-warmed growth media per well. No agar overlay is required since no secondary plaques are formed by VZV, which does not release virions into the overlaying medium.
    6. Monitor the cells for fluorescent foci formation. Usually, 5 days are sufficient for the foci to be easily countable, but this can vary between viruses and experiments.
    7. Using a 10x objective with a fluorescent microscope, count all the fluorescent foci (groups of fluorescent cells) in each well after 3-5 days. Take care not to count the same fluorescent focus twice, and screen all the wells in the same order. This process can be automated with a microscope equipped with a computerized stage and image analysis software. If all fluorescent foci have developed into plaques then units used for the final virus concentration are PFU.
    8. To calculate the PFU/ml, use the average number of foci in the wells with an input virus dilution resulting in 10-100 foci. For example, if the 1:25 dilution resulted in 25, 26 and 30 fluorescent foci, and the inoculate was 100 µl, then the stock concentration is


  3. Cell-free PRV and HSV1
    1. Grow Vero cells (green African monkey kidney cells) in a 24 well plate until they reach 90% confluence (~400,000 cells/well when seeding ~24 h before the infection). The use of Vero cells for HSV1 and PRV is documented in Moffat et al. (1998), Nicola and Straus (2004) and Pasdeloup et al. (2013).
    2. Prepare 10-fold serial dilutions of the virus in high glucose DMEM. Typically, the range of the dilutions is 10-2 to 10-7 of the initial stock. Keep the dilutions on ice.
    3. Inoculate the Vero cells with 100 µl/well of the virus in quadruplicates.
    4. Let the virus adsorb to the cell monolayer for 1 h in a CO2 incubator, agitating every 15 min.
    5. Remove the inoculum and cover the cells with 1 ml of overlaying medium containing 0.3% agarose per well. Work quickly so that the media will not start to solidify before its addition to the cells.
    6. Monitor the cultures for formation of fluorescent foci, usually less than 4 days.
    7. Using a 10x objective with a fluorescent microscope, count all the fluorescent foci (groups of fluorescent cells) in each well after 3-5 days. Take care not to count the same fluorescent focus twice, and screen all the wells in the same order. This process can be automated with a microscope equipped with a computerized stage and image analysis software. If all fluorescent foci have developed into plaques then units used for the final virus concentration are PFU.
    8. To calculate the PFU/ml, use the average number of foci in the wells with an input virus dilution resulting in 10-100 foci. For example, if the 10-4 dilution resulted in 40, 43 and 45 fluorescent foci per well, and the inoculated amount was 100 µl, then the stock concentration is

    9. If virus without fluorescent label are used, or it is desirable to count plaques rather than fluorescent loci, plaques are visualized by staining cultures with 0.1% crystal violet: Carefully lift the overlaying agar medium using a spatula, taking care to not disturb the cell monolayer. Fix the cells with 100 µl 4% PFA in PBS for 30 min at room temperature. Wash twice with PBS, and add 0.1% crystal violet solution for 10 min. Carefully wash with water until the liquid no longer has traces of blue (usually about 5 rinses).
    10. Count the plaques and calculate the PFU/ml as above.

  4. Multiplicity of infection (MOI) calculation
    1. In general, the equation of the multiplicity of infection is the following:

    2. In order to calculate the MOI, you should first determine the number of cells you are infecting, and the titer of the virus inoculated on them.
    3. For example, if 2.5 x 104 human foreskin fibroblasts are seeded in each well of a 24 well-plate, and 250 µl of 104 PFU/ml VZV were used to infect it, the MOI is

Representative data



Figure 1. Microscopic appearance of infectious foci using fluorescent virus. ARPE19 (A, B) or MeWo (C, D) cells were infected with cell-associated VZV-23GFP. Both fluorescence (A, C) and phase contrast (B, D) images of the same fields are shown. Scale bar =200 µm


Figure 2. Plaque assay for HSV1 gCGFP using crystal violet staining. 4 days after infection, cells were fixed and stained as described in the text. Viral concentration is decreasing from left to right. Crystal-violet stains the cells, which allows the visualization of the clear plaques. An arrow points to the dilution which yielded quantifiable plaques. Control, uninfected wells did not yield any detectable plaques (uninfected).

Recipes

  1. Fibroblast medium
    For 500 ml, combine 450 ml of high glucose DMEM with 50 ml fetal bovine serum. Supplement with 2.5 ml glutamine and 5 ml penicillin/streptomycin
  2. Overlaying media for free virus
    In advance, prepare a stock of 1.2% pure agarose for electrophoresis in PBS by autoclaving. Pre-heat the required amount of medium to 37 °C.
    Right before use, re-boil the agar in a microwave oven to liquefy it. Dilute the agar with the growth medium at a ratio of 1 agar volume to 3 medium volumes, e.g., add 6 ml agar to 18 ml medium in a 50 ml tube. This will result in 0.3% agar in medium which is sufficient to cover one 24-well plate.
    Keep the overlaying media tube inside a beaker with ~40 °C water, to prevent solidification.
  3. Crystal violet
    Dissolve 0.1% crystal violet in 10% ethanol (e.g., 5 ml ethanol and 45 ml H2O)
    First add the powder to the appropriate amount of 100% ethanol to completely dissolve it, and then add the required amount of water to generate 10% ethanol.

Acknowledgments

The HSV1 titration was based on an unpublished protocol of Dr. Maya Ilouze (Sheba Medical Center, Tel-HaShomer, Israel). We gratefully acknowledge the support of grant from the Israel Science Foundation 238/11 and a donation from the Maximillian Goode Foundation.

References

  1. Eisfeld, A. J., Yee, M. B., Erazo, A., Abendroth, A. and Kinchington, P. R. (2007). Downregulation of class I major histocompatibility complex surface expression by varicella-zoster virus involves open reading frame 66 protein kinase-dependent and -independent mechanisms. J Virol 81(17): 9034-9049.
  2. Heineman, T. C. and Cohen, J. I. (1994). Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro. J Virol 68(5): 3317-3323.
  3. Markus, A., Grigoryan, S., Sloutskin, A., Yee, M. B., Zhu, H., Yang, I. H., Thakor, N. V., Sarid, R., Kinchington, P. R. and Goldstein, R. S. (2011). Varicella-zoster virus (VZV) infection of neurons derived from human embryonic stem cells: direct demonstration of axonal infection, transport of VZV, and productive neuronal infection. J Virol 85(13): 6220-6233.
  4. Moffat, J. F., Zerboni, L., Kinchington, P. R., Grose, C., Kaneshima, H. and Arvin, A. M. (1998). Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol 72(2): 965-974.
  5. Nicola, A. V. and Straus, S. E. (2004). Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J Virol 78(14): 7508-7517.
  6. Pasdeloup, D., Labetoulle, M. and Rixon, F. J. (2013). Differing effects of herpes simplex virus 1 and pseudorabies virus infections on centrosomal function. J Virol 87(12): 7102-7112.
  7. Schmidt-Chanasit, J., Bleymehl, K., Rabenau, H. F., Ulrich, R. G., Cinatl, J., Jr. and Doerr, H. W. (2008). In vitro replication of varicella-zoster virus in human retinal pigment epithelial cells. J Clin Microbiol 46(6): 2122-2124.
  8. Sloutskin, A. and Goldstein, R. S. (2014). Laboratory preparation of Varicella-Zoster Virus: concentration of virus-containing supernatant, use of a debris fraction and magnetofection for consistent cell-free VZV infections. J Virol Methods 206: 128-132.

简介

病毒储液的滴定是在病毒的任何实验性使用之前的关键过程。 在这里我们介绍几种alphaherpesviruses的感染焦点测定法,基于原始斑块测定荧光标记病毒的滴定方法。 此外,提出了感染复数(MOI)的计算。

关键字:多重感染, 病毒感染, 疱疹病毒

材料和试剂

  1. 标准细胞系[例如 MeWo(ATCC HTB-65 TM ); ARPE19(ATCC CRL-2302 TM ); Vero(ATCC CCL-81 TM )]
  2. 荧光标记的病毒原种(遗传工程改造的VZV-66GFP,HSV1-gC-GFP和PRV-IE180-GFP由Paul R.Kinchington教授,匹兹堡大学匹兹堡大学赠送)
  3. 上述细胞的生长培养基(例如项目#6,成纤维细胞培养基)
    注意:但是,它可以是其他任何病毒和细胞对的媒介。
  4. 4%多聚甲醛(EMS 15710)在具有二价阳离子的磷酸盐缓冲盐水(例如Biological Industries,目录号:020201A)中。
  5. 高葡萄糖DMEM(Biological Industries,目录号:010551A)
  6. 胎牛血清(Biological Industries,目录号:041271A)
  7. 谷氨酰胺(Biological Industries,目录号:030201)
  8. 青霉素 - 链霉素(Biological Industries,目录号:030311)
  9. 琼脂糖低EEO(Hispanagar,目录号:D1500)
  10. 成纤维细胞培养基(参见配方)
  11. 覆盖媒体免费病毒(见配方)
  12. 结晶紫(Sigma-Aldrich,目录号:C0775)(参见Recipes)

设备

  1. 24孔组织培养板
  2. 培养箱37℃,5%CO 2
  3. 表光荧光倒置显微镜

程序

  1. 细胞相关水痘带状疱疹病毒(VZV)
    1. 在24孔板中生长MeWo或ARPE19细胞,直到它们达到90% 融合(约300,000或200,000个细胞/孔,接种前〜24小时   感染)。 使用500微升/孔体积生长和感染 细胞。 在Heineman和Cohen中记载了MeWo细胞用于VZV的用途   (1994),Eisfeld等人(2007)和Markus等人(2011)。
    2. 准备 10倍系列稀释的VZV感染的MeWo细胞。 通常, 稀释度范围为初始原料的10 -1 -10至10 -6 -6 。 音量 接种到每个孔可以高达500微升。
    3. 添加感染   细胞至含有90%汇合的初始细胞的24孔板 培养物一式三份,并在37℃孵育至少2小时。 在添加后没有特殊的需要更换介质   接种物,因此感染的细胞也可以留在过夜。 不需要琼脂覆盖,因为没有由VZV形成二次噬菌斑, 其不会将病毒体释放到覆盖介质中
    4. 使用a   10x物镜用荧光显微镜,计数所有荧光 焦点(荧光细胞组)在2-5天后。 采取 注意不要计数相同的荧光对焦两次,并且屏幕全部 井顺序相同。 这个过程可以用显微镜自动化   配备了计算机化的舞台和图像分析软件。 我摔倒 荧光焦点已经发展成斑块,然后用于的单位 最终病毒浓度是噬菌斑形成单位(PFU)
    5. 至 计算PFU/ml,使用孔中的平均焦点数 输入病毒稀释导致10-100个灶。 例如,如果 10 稀释导致每孔24,26和30个荧光病灶 接种量为100μl,则库存浓度为


  2. 无细胞VZV
    1. 在24孔板中生长ARPE19细胞,直到它们达到90%汇合 (在接种前约〜200,000个细胞/孔〜感染前〜24小时)。 这些 细胞在较低VZV浓度下更容易感染 MeWo或MRC5细胞(Schmidt-Chanasit等人,2008; Pasdeloup等人, 2013)。 使用500μl/孔生长和感染细胞
    2. 准备5倍系列稀释的无细胞VZV在高葡萄糖DMEM。 在我们的经验中,稀释病毒在生长培养基中的细胞 对血清的存在不敏感似乎不影响病毒   感染性。 通常,稀释度的范围为1:5至1:625 初始库存。
    3. 用100-150μl/孔的病毒稀释液接种ARPE19细胞一式三份。
    4. 在CO 2培养箱中,让病毒吸附到细胞单层2小时,偶尔搅动。
    5. 取出接种物,每孔加入0.5ml预热的生长培养基。   不需要琼脂覆盖层,因为没有形成二次噬菌斑 VZV,其不会将病毒体释放到覆盖培养基中
    6. 监测细胞的荧光灶形成。 通常,5天是 足以使焦点容易计数,但这可以变化 病毒和实验之间
    7. 使用10x物镜 荧光显微镜,计数所有荧光灶(组 荧光细胞)3-5天后。 小心不要计数 相同的荧光焦点两次,并筛选所有的孔在相同 订购。 这个过程可以用配备有a的显微镜自动化 计算机化阶段和图像分析软件。 如果所有荧光焦点 已经发展成为用于最终病毒的单位然后单位 浓度为PFU
    8. 要计算PFU/ml,请使用平均值 在输入病毒稀释导致的孔中的病灶数目 10-100灶。 例如,如果1:25稀释导致25,26和 30荧光灶,接种量为100μl,然后加入母液 浓度为


  3. 无细胞PRV和HSV1
    1. 在24孔板中生长Vero细胞(绿色非洲猴肾细胞) 直到它们达到90%汇合(〜400,000个细胞/孔,接种〜24小时 感染前)。 Vero细胞用于HSV1和PRV的用途是 记载于Moffat等人(1998),Nicola和Straus(2004)和 Pasdeloup (2013)。
    2. 准备10倍连续稀释的 病毒在高葡萄糖DMEM。 通常,稀释的范围是 10 -2 至10 -7 。 将稀释液置于冰上。
    3. 接种100微升/孔的病毒一式四份的Vero细胞。
    4. 让病毒在CO 2培养箱中吸附细胞单层1小时,每15分钟搅动一次。
    5. 取出接种物并用1ml覆盖物覆盖细胞 每孔含有0.3%琼脂糖的培养基。 快速工作,使媒体 在加入细胞之前不会开始凝固
    6. 监测培养物的荧光灶的形成,通常少于4天。
    7. 使用10倍物镜用荧光显微镜,计数所有 荧光灶(荧光细胞组)3-5天后每孔 天。注意不要计数相同的荧光对焦两次,和 以相同的顺序筛选所有孔。这个过程可以自动化 用装备有计算机化阶段和图像分析的显微镜 软件。如果所有荧光焦点已经发展成斑块然后单位  用于最终病毒浓缩的是PFU
    8. 计算  PFU/ml,使用输入病毒的孔中的平均焦点数  稀释导致10-100个灶。例如,如果10 -4 稀释 导致每孔40,43和45个荧光灶,并接种 量为100μl,则库存浓度为

    9. 如果病毒 没有使用荧光标记,或者期望计数噬菌斑 而不是荧光位点,通过染色显示噬斑 含有0.1%结晶紫的培养物:小心地提起覆盖的琼脂 培养基,使用刮刀,注意不打扰细胞单层。 固定细胞与100微升4%PFA的PBS在室温下30分钟。 用PBS洗涤两次,并加入0.1%结晶紫溶液10分钟。 小心用水洗涤,直到液体不再有蓝色的痕迹 (通常约5次冲洗)。
    10. 计数噬菌斑并如上计算PFU/ml。

  4. 感染复数(MOI)计算
    1. 一般来说,感染复数的公式如下:

    2. 为了计算MOI,你应该首先确定数字   的感染细胞和接种病毒的滴度 他们
    3. 例如,如果2.5×10 4个人包皮成纤维细胞 接种在24孔板的每个孔中,加入250μl10 4 PFU/ml VZV 用来感染它,MOI是

代表数据



图1.使用荧光病毒的感染灶的显微外观。用细胞相关的VZV-23GFP感染ARPE19(A,B)或MeWo(C,D)细胞。示出了相同场的荧光(A,C)和相位对比(B,D)图像。比例尺=200μm


图2.使用结晶紫染色的HSV1gCGFP的噬斑测定。感染后4天,如本文所述固定细胞和染色。病毒浓度从左到右降低。结晶紫染色细胞,这允许清楚的斑块的可视化。箭头指向产生可量化噬斑的稀释。对照,未感染的孔不产生任何可检测的斑块(未感染)。

食谱

  1. 成纤维细胞培养基
    对于500ml,将450ml高葡萄糖DMEM与50ml胎牛血清组合。补充2.5ml谷氨酰胺和5ml青霉素/链霉素
  2. 覆盖媒体免费病毒
    事先,准备1.2%纯的琼脂糖的储备液,通过高压灭菌法在PBS中电泳。将所需量的培养基预热至37℃。
    在使用前,将琼脂在微波炉中再煮沸以液化。用生长培养基以1琼脂体积与3培养基体积的比例稀释琼脂,例如在50ml管中将6ml琼脂加入18ml培养基中。这将导致在培养基中的0.3%琼脂,其足以覆盖一个24孔板。
    将覆盖介质管放在约40°C水的烧杯中,以防止凝固
  3. 水晶紫
    将0.1%结晶紫溶解在10%乙醇(例如,5ml乙醇和45ml H 2 O)中。
    首先将粉末加入适量的100%乙醇中使其完全溶解,然后加入所需量的水生成10%乙醇。

致谢

HSV1滴定基于Maya Ilouze博士(Sheba Medical Center,Tel-HaShomer,Israel)的未发表的方案。我们衷心感谢以色列科学基金会238/11的赠款和马克西米利亚古德基金会的捐赠。

参考文献

  1. Eisfeld,A.J.,Yee,M.B.,Erazo,A.,Abendroth,A.and Kinchington,P.R。(2007)。 水痘带状疱疹病毒对I类主要组织相容性复合物表面表达的下调涉及开放阅读框架66蛋白激酶 - 依赖和独立的机制。 Virol 81(17):9034-9049。
  2. Heineman,T.C。和Cohen,J.I。(1994)。 核糖核酸还原酶水痘带状疱疹病毒大亚基的缺失会损害病毒在体外的生长。 J Virol 68(5):3317-3323。
  3. Markus,A.,Grigoryan,S.,Sloutskin,A.,Yee,M.B.,Zhu,H.,Yang,I.H.,Thakor,N.V.,Sarid,R.,Kinchington,P.R.and Goldstein, 源自人类胚胎干细胞的神经元的水痘带状疱疹病毒(VZV)感染:轴突的直接证明感染,VZV的转运和生产性神经元感染。 J Virol 85(13):6220-6233。
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引用:Sloutskin, A. and Goldstein, R. S. (2014). Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses. Bio-protocol 4(22): e1295. DOI: 10.21769/BioProtoc.1295.
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