Enterovirus Competition Assay to Assess Replication Fitness

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Nature Microbiology
Nov 2018



In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.

Keywords: Enterovirus (肠病毒), Competition assay (竞争实验), Virus titration (病毒滴定), Dual infection (双重感染), Pairwise growth competition assay (成对增长竞争测定法), Virus passaging (病毒传代), Sanger sequencing (Sanger 测序)


Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. In our recent paper (Lulla et al., 2019) we reported that many enterovirus genomes harbor an upstream open reading frame (uORF) that encodes an additional viral protein, UP (upstream protein). Using the echovirus 7 (EV7) reverse genetics system, we created two UP knock-out mutants (EV7-Loop and EV7-PTC), which exhibited wtEV7-like growth properties. Therefore, we decided to perform a competition assay to determine possible subtle fitness defects.

Materials and Reagents

  1. Materials
    1. Pipette tips (Fisher, catalog numbers: 02-707-426, 02-707-403, 02-707-438)
    2. 1.5 ml microfuge tubes (STARLAB, catalog number: 51615-5500)
    3. T175 tissue culture flasks (TPP® tissue culture flasks, Sigma, catalog number: Z707562)
    4. 12-well polystyrene culture plates (TPP® tissue culture plates, Sigma, catalog number: Z707775)

  2. Viruses
    Echovirus 7 (EV7), derived from EV7 infectious clone
    Note: The cDNA of Echovirus 7 strain Wallace was sourced from Michael Lindberg (GenBank accession number AF465516, with the silent substitution 1687G-to-A) and was cloned downstream of a T7 RNA promoter. Mutant viruses EV7-Loop and EV7-PTC were generated by mutagenesis of the original EV7 infectious clone and prepared exactly as wt EV7 (Lulla et al., 2019).

  3. Cell lines
    RD cells (human rhabdomyosarcoma cell line, ATCC, CCL-136), verified mycoplasma-free by next-generation sequencing

  4. Reagents
    1. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, catalog number: D6546)
    2. Fetal bovine serum (FBS) (PAN Biotech, catalog number: P40-37500)
    3. Penicillin-streptomycin (10,000 U/ml) (Life Technologies, catalog number: 15140-122)
    4. 200 mM L-Glutamine (Gibco, catalog number: 25030081)
    5. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 14190-144)
    6. 1 M HEPES buffer, pH range: 7.2-7.5 (Life Technologies, catalog number: 15630-080)
    7. 0.25% Trypsin (Gibco, catalog number: 15090046-100)
    8. Bovine serum albumin (BSA) (PAN Biotech, catalog number: P06-1395500)
    9. Direct-zolTM RNA MiniPrep Plus (Zymo Research, catalog number: R2072) 
    10. PhusionTM RT-PCR Kit (Thermo Scientific, catalog number: F-546S)
    11. Complete DMEM (see Recipes)
    12. Serum-free DMEM (see Recipes)


  1. -80 °C freezer
  2. Vortexer
  3. Pipettes: 1000 µl, 200 µl, 20 µl, 2 µl
  4. CO2 incubator
  5. Haemocytometer
  6. BSL2 cell culture cabinet
  7. Rocking platform shaker
  8. Phase-contrast inverted microscope (Nikon TMS)
  9. 4 °C refrigerator
  10. Autoclave


  1. BioEdit (Free software, Ibis Therapeutics, http://www.mbio.ncsu.edu/BioEdit/bioedit.html)


IMPORTANT NOTE: All experiments with viruses (i.e., all pre-Trizol RNA isolation steps) should be performed inside a biosafety level 2 (BSL2) tissue culture laboratory according to the country and institution regulations and required permits regarding enterovirus strains handling and storage.

  1. Infection and virus passaging
    1. Prepare cells for infection 1 day before by seeding 3 x 105 RD cells per well on 12-well plates (the aim is to achieve 5 x 105 RD cells per well by the next day, which corresponds to subconfluent RD monolayers).
      1. RD cells seeded on T175 flasks and grown in complete DMEM until 80-90% confluency. Aspirate media from the cells and rinse with 10 ml of PBS. Trypsinize RD cells using 3 ml of 0.25% trypsin by gently tapping the flask.
      2. Once cells are detached, add 10 ml of complete DMEM, ensure the cells are properly resuspended by pipetting up and down. Count cells in suspension using hemocytometer.
      3. Dilute cell suspension with complete DMEM to get concentration corresponding to 3 x 105 cells per 1 ml. Seed 1 ml of cell suspension to each well of 12-well plates.
      4. Incubate overnight in 37 °C, 5% CO2 incubator.
    2. Check the cell monolayers using a microscope (equal healthy cell monolayers, no CPE). Remove media from cell monolayers and infect with a total multiplicity of infection (MOI) of 0.1, in 0.2 ml of serum-free DMEM. Use the following formula to calculate the amount of virus needed:

      Example: For 109 PFU/ml virus stock use, 5 x 105 cells x 0.1 PFU/cell x 103 μl/ml/109 PFU/ml = 0.05 μl.
    3. Mix using wt EV7 and mutant at either equal or 1:9 proportions, with a total MOI 0.1.
      Note: This assay can be completed with other viruses and cell lines. Different proportions and MOI can be used for this experiment depending on the virus growth kinetics and susceptibility of cells for infection. Using MOI 0.1 means that more than 90% of the cells remain uninfected providing enough space for competing viruses; any growth difference should also accumulate over passaging. 
    4. Use triplicates for each infection, monoinfections for positive controls and mock (uninfected) cells as negative controls as suggested in Figure 1.

      Figure 1. Sample layout of two 12-well plates at either equal (Left) or 1:9 (Right) proportions. Every subsequent passage is performed on similarly aranged 12-well plates.

    5. Incubate infected cells at room temperature on a gently rocking platform for 1 h. To remove unbound virus, aspirate infection media, wash each well with 1 ml of serum-free DMEM, and overlay cells with 0.5 ml of serum-free DMEM.
    6. Incubate at 37 °C in a CO2 incubator overnight (16-20 h) until complete CPE is observed.
    7. Collect media from infected plates to a 1.5 ml tube, centrifuge at 9,600 x g for 5 min, then transfer to the new 1.5 ml tube and measure the volume.
    8. Add 0.2% BSA to supernatant (10 μl of 10% BSA per 0.5 ml of clarified supernatant).
    9. Perform 5 blind passages (with estimated, but unknown titer) using 1:10,000 volume of obtained virus stock (corresponds to MOI 0.05-0.2). Use the same infection protocol as before (steps 1-8).
      Note: For less efficiently replicating viruses more than 5 passages could be needed.
    10. Store virus stocks at -80 °C.
    11. Add 1 ml PBS to remaining cells and collect them by pipetting into a 1.5 ml tube (infected cells detach easily and do not require scraping). Centrifuge cells at 9,600 x g for 5 min, remove supernatant and proceed to RNA isolation from the infected cells.

  2. RNA isolation from infected cells
    1. Isolate RNA from passages 1 and 5 using Direct-zolTM RNA MiniPrep Plus (Zymo Research) according to the manufacturer’s protocol (Direct-zolTM RNA MiniPrep Plus INSTRUCTION MANUAL ver.1.10.1).
    2. Perform RT-PCR using Thermo ScientificTM PhusionTM RT-PCR Kit using virus-specific primers and manufacturer’s recommendations (Thermo Scientific Phusion RT-PCR Kit manual). The example design of the introduced mutations and sequencing primer is presented in Figure 2.

      Figure 2. Example design of mutant virus genomes and sequencing primer. Depending on sequencing facilities, a 300-1000 bp long PCR fragment should be optimal for this assay.

    3. Perform Sanger sequencing of the PCR fragment containing the mutated region of the virus genome.
      Note: Please consider the nature of the virus (intracellular or budded virions) for choosing the correct sample to analyze. Sometimes analyzing RNA from media rather than from infected cells can be considered. For enteroviruses it is not a crucial step; therefore an easier approach (RNA isolation from infected cells) was chosen.

Data analysis

Compare chromatograms and evaluate based on three RT-PCR products from each analyzed sample (Figure 3). In the experimental conditions tested viruses do not differ in their growth properties. The chromatograms should shift if the viruses differ in their growth properties. Compare the peak areas from at least three different chromatograms for each sample and estimate the changes. If wt virus out-competes the mutant, the chromatogram of the fifth passage sample should match the wt virus chromatogram.

Figure 3. Sample sequencing chromatograms of RT-PCR products for the competition. Experiment of EV7, EV7-PTC and EV7-Loop mutants at initial infection (P1) and after five passages (P5). The figure is reproduced from Supplementary Figure 3 in Lulla et al. (2019).


  1. Complete DMEM
    500 ml DMEM
    50 ml FBS
    5 ml 200 mM L-Glutamine
    5 ml penicillin-streptomycin (10,000 U/ml)
    10 ml 1 M HEPES pH 7.2-7.5
    Store at 4 °C
  2. Serum-free DMEM
    500 ml DMEM
    5 ml 200 mM L-Glutamine
    5 ml penicillin-streptomycin (10,000 U/ml)
    10 ml 1 M HEPES, pH 7.2-7.5
    Store at 4 °C


This work was supported by Wellcome Trust grant [106207] and European Research Council grant [646891] to A.E.F. The cDNA of Echovirus 7 strain Wallace was sourced from Michael Lindberg. We thank Hazel Stewart for critical reading of the protocol.

Competing interests

The authors declare that they do not have any conflicts of interests or competing interests.


  1. Lulla, V., Dinan, A. M., Hosmillo, M., Chaudhry, Y., Sherry, L., Irigoyen, N., Nayak, K. M., Stonehouse, N. J., Zilbauer, M., Goodfellow, I. and Firth, A. E. (2019). An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells. Nat Microbiol 4(2): 280-292.


摘要:在病毒学中,两种病毒的适合度之间的差异可以通过使用各种方法来确定,例如病毒滴度,生长曲线分析,病毒感染性的测量,产生的RNA拷贝的分析和病毒蛋白质产生。但是,对于密切执行的病毒,通常很难区分这些差异。 体外竞争分析是确定在细胞培养中复制的许多病毒的病毒复制适应性的敏感工具。通常从多循环生长竞争测定法测量相对病毒复制适应性。竞争测定提供了对病毒适应性的敏感测量,因为病毒在相同的生长条件下竞争细胞靶标。该方案描述了肠道病毒的竞争测定,并且包含两种初始感染的替代形式,其可以根据每个特定实验的具体目标而变化。该方案涉及用竞争病毒感染细胞,传代,从感染细胞中提取RNA,RT-PCR和Sanger测序,然后比较分析在各种初始感染条件下获得的所得色谱图。该技术适用于许多病毒家族的成员,例如甲病毒,黄病毒,瘟病毒和具有已建立的反向遗传系统的其他RNA病毒。

背景:肠道病毒包括大量哺乳动物病原体,包括脊髓灰质炎病毒。 人类的病理学范围从亚临床到急性弛缓性麻痹,心肌炎和脑膜炎。 在我们最近的论文(Lulla et al。,2019)中,我们报道了许多肠道病毒基因组具有上游开放阅读框(uORF),其编码另外的病毒蛋白UP(上游蛋白)。 使用埃可病毒7(EV7)反向遗传学系统,我们创建了两个UP敲除突变体(EV7-Loop和EV7-PTC),它们表现出类似wtEV7的生长特性。 因此,我们决定进行竞争测定以确定可能的微妙健身缺陷。

关键字:肠病毒, 竞争实验, 病毒滴定, 双重感染, 成对增长竞争测定法, 病毒传代, Sanger 测序


  1. 移液器吸头(Fisher,目录号:02-707-426,02-707-403,02-707-438)
  2. 1.5 ml微量离心管(STARLAB,目录号:51615-5500)
  3. T175组织培养瓶(TPP ®组织培养瓶,Sigma,目录号:Z707562)
  4. 12孔聚苯乙烯培养板(TPP ®组织培养板,Sigma,目录号:Z707775)

  • 病毒
    注意:Echovirus 7菌株Wallace的cDNA来自Michael Lindberg(GenBank登录号AF465516,沉默取代 1687 G-to-A)并克隆到T7 RNA启动子的下游。突变病毒EV7-Loop和EV7-PTC通过原始EV7感染性克隆的诱变产生,并且完全按重量EV7制备(Lulla 等 ,2019)。

  • 细胞系

  • 试剂
    1. Dulbecco的改良Eagle's培养基(DMEM)(Sigma,目录号:D6546)
    2. 胎牛血清(FBS)(PAN Biotech,目录号:P40-37500)
    3. 青霉素 - 链霉素(10,000 U / ml)(Life Technologies,目录号:15140-122)
    4. 200 mM L-谷氨酰胺(Gibco,目录号:25030081)
    5. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14190-144)
    6. 1 M HEPES缓冲液,pH范围:7.2-7.5(Life Technologies,目录号:15630-080)
    7. 0.25%胰蛋白酶(Gibco,目录号:15090046-100)
    8. 牛血清白蛋白(BSA)(PAN Biotech,目录号:P06-1395500)
    9. Direct-zol TM RNA MiniPrep Plus(Zymo Research,目录号:R2072) 
    10. Phusion TM RT-PCR试剂盒(Thermo Scientific,目录号:F-546S)
    11. 完整的DMEM(见食谱)
    12. 无血清DMEM(见食谱)
  • 设备

    1. -80°C冰箱
    2. 涡流混合器
    3. 移液管:1000μl,200μl,20μl,2μl
    4. CO 2 培养箱
    5. 血球
    6. BSL2细胞培养柜
    7. 摇摆平台振动筛
    8. 相位倒置显微镜(尼康TMS)
    9. 4°C冰箱
    10. 高压灭菌器


    1. BioEdit(免费软件,Ibis Therapeutics, http://www.mbio.ncsu.edu/BioEdit /bioedit.html


    重要注意事项:所有病毒实验(即所有前Trizol RNA分离步骤)均应在生物安全2级(BSL2)组织培养实验室内根据国家和机构法规进行,并且需要有关肠道病毒株处理的许可证。存储。

    1. 感染和病毒传代
      1. 在前一天通过在12孔板上每孔接种3×10 5 RD细胞来制备感染细胞(目的是每孔达到5×10 5 RD细胞。到第二天,这对应于亚汇合的RD单分子层)。
        1. 将RD细胞接种在T175烧瓶上并在完全DMEM中生长直至80-90%汇合。从细胞中吸出培养基并用10ml PBS冲洗。通过轻轻敲击烧瓶,使用3ml 0.25%胰蛋白酶胰蛋白酶消化RD细胞。
        2. 一旦细胞分离,加入10毫升完全DMEM,确保通过上下移液正确重悬细胞。使用血细胞计数器计数悬浮细胞。
        3. 用完全DMEM稀释细胞悬浮液,得到相当于每1ml 3×10 5个细胞的浓度。将1ml细胞悬浮液接种到12孔板的每个孔中。
        4. 在37℃,5%CO 2 培养箱中孵育过夜。
      2. 使用显微镜检查细胞单层(相同的健康细胞单层,无CPE)。从细胞单层中除去培养基,并在0.2ml无血清DMEM中以0.1的总感染复数(MOI)感染。使用以下公式计算所需的病毒量:

        示例:对于10 9 PFU / ml病毒库存使用,5 x 10 5 细胞x 0.1 PFU /细胞x 10 3 μl/ ml / 10 9 PFU / ml =0.05μl。
      3. 使用wt EV7和突变体以相等或1:9的比例混合,总MOI为0.1。
        注意:此测定可以与其他病毒和细胞系一起完成。取决于病毒生长动力学和细胞对感染的易感性,不同比例和MOI可用于该实验。使用MOI 0.1意味着超过90%的细胞保持未感染,为竞争病毒提供足够的空间;任何增长差异也应累积在传代上。 
      4. 每次感染使用一式三份,阳性对照单次感染和模拟(未感染)细胞作为阴性对照,如图1所示。


      5. 将感染的细胞在室温下在轻轻摇动的平台上孵育1小时。为了去除未结合的病毒,吸出感染培养基,用1ml无血清DMEM洗涤每个孔,并用0.5ml无血清DMEM覆盖细胞。
      6. 在37℃下在CO 2 培养箱中孵育过夜(16-20小时)直至观察到完全CPE。
      7. 将感染的培养板中的培养基收集到1.5ml管中,以9,600 x g 离心5分钟,然后转移到新的1.5ml管中并测量体积。
      8. 向上清液中加入0.2%BSA(每0.5ml澄清上清液10μl10%BSA)。
      9. 使用1:10,000体积的获得的病毒原种(对应于MOI 0.05-0.2)进行5次盲传(具有估计但未知的滴度)。使用与以前相同的感染协议(步骤1-8)。
      10. 将病毒储存在-80°C。
      11. 向剩余的细胞中加入1ml PBS,并通过移液到1.5ml管中收集它们(感染的细胞容易分离并且不需要刮擦)。在9,600 x g 离心细胞5分钟,去除上清液并从感染细胞中进行RNA分离。

    2. 从感染细胞中分离RNA
      1. 根据制造商的方案使用Direct-zol TM RNA MiniPrep Plus(Zymo Research)从第1代和第5代分离RNA( Direct-zol TM RNA MiniPrep Plus INSTRUCTION MANUAL ver.1.10.1 )。
      2. 使用Thermo Scientific TM Phusion TM RT-PCR试剂盒,使用病毒特异性引物和制造商的建议进行RT-PCR( Thermo Scientific Phusion RT-PCR Kit手册)。引入的突变和测序引物的示例设计如图2所示。

        图2.突变病毒基因组和测序引物的示例设计。根据测序设施,300-1000 bp长PCR片段应该是此测定的最佳选择。

      3. 对含有病毒基因组突变区域的PCR片段进行Sanger测序。



    图3.用于竞争的RT-PCR产物的样品测序色谱图。 EV7,EV7-PTC和EV7-Loop突变体在初始感染(P1)和五次传代后的实验(P5)。该图从Lulla 等人的补充图3中再现(2019)。


    1. 完成DMEM
      5毫升青霉素 - 链霉素(10,000 U / ml)
      10毫升1M HEPES pH 7.2-7.5
    2. 无血清DMEM
      5毫升青霉素 - 链霉素(10,000 U / ml)
      10毫升1M HEPES,pH 7.2-7.5


    这项工作得到了Wellcome Trust拨款[106207]和欧洲研究理事会授予[646891] A.E.F.的支持。 Echovirus 7菌株Wallace的cDNA来自Michael Lindberg。我们感谢Hazel Stewart对协议的批判性阅读。




    1. Lulla,V.,Dinan,AM,Hosmillo,M.,Chaudhry,Y.,Sherry,L.,Irigoyen,N.,Nayak,KM,Stonehouse,NJ,Zilbauer,M.,Goodfellow,I。和Firth,AE(2019)。 肠道病毒的上游蛋白质编码区调节肠道上皮细胞中的病毒感染。 Nat Microbiol 4(2):280-292。
    • English
    • 中文翻译
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    Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Lulla, V. and Firth, A. E. (2019). Enterovirus Competition Assay to Assess Replication Fitness. Bio-protocol 9(10): e3233. DOI: 10.21769/BioProtoc.3233.