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In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

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Antimicrobial Agents and Chemotherapy
Dec 2015



In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of EV-D68 infections. In order to evaluate the antiviral activity of potential inhibitors of EV-D68 replication, a cell-based cytopathic effect (CPE) reduction assay was developed (Sun et al., 2015).

Keywords: Enterovirus D68 (肠道病毒D68), Antiviral assay (抗病毒测定), CPE (CPE), MTS/PMS solution (MTS/PMS溶液), Cell culture (细胞培养)


As a re-emerging pathogen, antiviral compounds targeting EV-D68 were rarely reported before. It is urgently needed to establish and develop antiviral methods to combat the potential EV-D68 epidemic. Here, we report a detailed protocol which can be used to identify selective anti-EV-D68 compounds. To screen and identify potential antiviral compounds, the MTS-based CPE reduction assay is reproducible, easy-to-use and time-saving method which is widely used. This method relies on the ability of a compound to inhibit EV-D68-induced reduction of CPE. When a compound actively inhibits the replication of EV-D68, the virus-induced CPE will be reduced or absent. As a consequence, the metabolically active cells are able to convert a yellow tetrazolium salt substrate (MTS/PMS) to a brown-colored formazan product. When a compound does not have antiviral effects, the host cells die of virus-induced CPE, which will lack metabolic activity, and the yellow substrate remains un-metabolized. The colorimetric conversion is quantitative, as an EC50 can be calculated from the data generated by this assay.

Materials and Reagents

  1. Centrifuge tube, 14 ml (Corning, Falcon®, catalog number: 352059 )
  2. 1.5 ml Eppendorf Snap-Cap microcentrifuge tubes (VWR, catalog number: 21008-959 )
  3. 4 ml Screw neck vials, amber glass (VWR, catalog number: 548-0052 )
  4. Transparent 96-well (flat-bottom) tissue culture plates (Corning, Falcon®, catalog number: 353072 )
  5. Tissue culture flasks, 150 cm2 (TPP, catalog number: 90856 )
  6. Pipette tips (10 µl, 100 µl, 1,000 µl)
  7. Disposable serological pipets (5 ml, 10 ml and 25 ml)
  8. Reagent reservoir, 25 ml (Biotix, catalog number: SR-0025-5SWM )
  9. Nalgene rapid-flow sterile disposable filter units with SFCA membrane (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 156-4020 )
  10. Hela Rh cell line (a Hela subclone, highly susceptible and permissible to rhinovirus-induced CPE, a gift from Janssen Pharmaceutica, Belgium) (Lacroix et al., 2014)
  11. All EV-68 strains were cultivated on HeLa Rh cells and stored at -80 °C
    1. EV-D68 strains (742, 947, 2042, 1348, 2284 and 670) were isolated from the Netherlands (a kind of gift from Adam Meijer, Diagnostics and Screening National Institute for Public Health and the Environment [RIVM], Bilthoven)
    2. EV-D68 strains (US/KY/14-18953, US/IL/14-18952 and US/MO14-18947) – these strains were originally obtained from BEI Resources
    Note: The lysate of all virus strains were used for antiviral assay, and virus titer was measured by endpoint titration assay.
  12. Dulbecco’s phosphate-buffered saline (DPBS), no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  13. 0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
  14. Minimum essential media (MEM) Rega-3 (Thermo Fisher Scientific, GibcoTM, catalog number: 19993013 )
  15. Potential antiviral compounds
    1. SG85*
    2. Pleconaril*
    3. Vapendavir*
    4. Pirodavir*
    5. Rupintrivir*
    6. Enviroxime*
    7. Favipiravir*
    Note: All *compounds were dissolved in DMSO at a concentration of 10 mg/ml and stored in screw neck vials at 4 or -20 °C.
  16. Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH30084.03 )
  17. L-glutamine 200 mM (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  18. Sodium bicarbonate 7.5% solution (Thermo Fisher Scientific, GibcoTM, catalog number: 25080060 )
  19. Minimum essential media (MEM), no glutamine, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 51200038 )
  20. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  21. CellTiter 96® AQueous MTS reagent powder (Promega, catalog number: G1111 )
  22. HCl
  23. Dimethyl sulfoxide (VWR, catalog number: 67-68-5 )
  24. Growth culture medium (see Recipes)
  25. Assay medium (see Recipes)
  26. MTS/PMS solution (see Recipes)


  1. Pipette controller, PIPETBOY acu2 (VWR, catalog number: 612-0927 )
  2. Multichannel pipette, 10-100 µl (Eppendorf)
  3. Incubator at 37 °C (and 35 °C ) and 5% CO2 (Binder)
  4. Mid bench centrifuge (Sigma Laborzentrifugen, mode: 4K15C )
  5. MoxiTM Z Mini Automated Cell Counter (ORFLO Technologies, catalog number: MXZ001 )
  6. SpectraMax 190 microplate reader (Molecular Devices, model: SpectraMax 190 )
  7. pH bench meter (Xylem Analytics, WTW, model: inoLab® 9310 IDS )
  8. Inverted microscope (Motic, model: AE21 )
  9. Safety cabinet (Telstar)


  1. GraphPad Prism


  1. Grow Hela Rh cells to confluence in growth culture medium (see Recipes) at 37 °C and 5% CO2.
    Note: Hela Rh cells are passaged by 1 in 10, and they will be confluence after 3-4 days.
  2. Aspirate the supernatant, wash the cells three times with 10 ml PBS and detach the cells using trypsin at 37 °C about 3-5 min.
  3. When the cells are detached, collect the cells with 10 ml growth medium in a 14 ml tube. Centrifuge the cells for 6 min at 269 x g at room temperature.
  4. Re-suspend the cell pellet in 10 ml assay medium (see Recipes).
  5. Count the number of cells. Seed the cells into a 96-well plate at a density of 15,000 cells in 100 µl assay medium per well. Incubate the cells for 24 h in the incubator at 37 °C and 5% CO2 to allow the cells to adhere to the plate.
  6. Dilute the compounds to the desired start concentration in assay medium. This start concentration should be 6x the desired final concentration to prepare for the 1-in-3 serial dilution. Add 50 µl of the 6x compound dilution to the 100 µl assay medium in the second column, wells B2 to G2 which seeded with cells (a maximum of 6 compounds, Figure 1).

    Figure 1. Layout of 96-well plate for antiviral assay. Grey wells only contain medium; VC: virus control, CC: cell control.

  7. Mix the medium of column 2 by gentle pipetting and transfer 50 µl of medium from this column to the next column 3 (B3-G3) (see Figure 2A); repeat this up to column 9 (B9-G9). Discard 50 µl of medium from column 9. Column 10 (B10-G10) will be the virus control (VC, assay medium + EV68) and column 11 (B11-G11) will be the cell control (CC, only assay medium).
  8. Add 100 µl of EV68 of the desired dilution (MOI = 0.001) to column 2-10 and 100 µl assay medium to column 11 (see Figure 2B). Wells on the edge of the 96-well plate (see Figure 1, labelled in grey) contain 200 µl medium but will not be used because edge effects may influence the assay results.

    Figure 2. Workflow. A. Gently mix the assay medium in the treated and infected wells. Transfer 50 µl of medium to the next well to prepare a 1-in-3 dilution series. B. Following the preparation of compound dilutions, add 100 µl of assay medium containing 2x virus dilution.

  9. Place the 96-well plate in the incubator (35 °C, 5% CO2 and 95% relative humidity) for 3-4 days until complete virus-induced cytopathic effect (CPE) is observed by microscope in the VC wells (see Figure 3).

    Figure 3. HeLa Rh cells in cell control wells (A) and virus control wells (B). Scale bars = 35 μm.

  10. Remove the assay medium of all wells in column 2-11 and add 100 µl of 5% MTS/PMS dilution in phenol red- and glutamine-free medium.
  11. Following incubation for 40-60 min in the 37 °C 5% CO2 incubator, measure the absorbance in each well of the 96-well plate with a microplate reader at a wavelength of 498 nm.

Data analysis

  1. The antiviral activity is calculated as follows:
    % antiviral activity = (ODEV68+Compound - ODVC)/(ODCC - ODVC) x 100.
    ODCC and ODVC represent average values of optical density (OD) of the cell control (column 11) and the virus control (column 10), respectively,
    ODEV68+Compound corresponds to the OD value of each infected and treated well.
  2. Calculate the EC50 value of each compound, which is defined as the concentration of compound at which the virus-induced CPE is reduced by 50%. This value of antiviral activity is calculated by curve fitting which was generated from GraphPad Prism.
    Note: Alternatively, logarithmic interpolation can be used to estimate EC50 values if GraphPad Prism is not available. The below Representative data is shown by logarithmic interpolation.

Representative data

  1. % Antiviral activity of each concentration is calculated and showed below:
    SG85 (µM)
    % antiviral activity

    EC50 =10((Log(A) – Log(B)) x ((C - 50)/(CD)) + Log(B))
    A: compound concentration at which % antiviral activity is < 50%
    B: compound concentration at which % antiviral activity is > 50%
    C: value of % antiviral activity > 50%
    D: value of % antiviral activity < 50%
  2. An example of a dose-response curve of the antiviral activity of the protease inhibitor SG85 against different EV68 clinical isolates (see Figure 4). EC50 values are presented in Sun et al. (2015).

    Figure 4. The antiviral activity of SG85 against EV68 clinical isolates was determined in a CPE reduction assay with an MTS/PMS readout


  1. Depending on the cell type used in the antiviral assay, the amount of cells per well may need to be optimized. It is important that cells in the cell control group do not over-grow after 3 or 4 days.
  2. Use the lowest viral inoculation that still results in 100% full CPE in all wells at day 3 or 4 post infection.
  3. If a 1-to-2 serial dilution of compound is preferred, prepare a 4x concentrated compound dilution and add 100 µl of this compound dilution to column 2; Then transfer 100 µl from well to well to generate the 1:2 dilution series.
  4. Because of its low optimum growth temperature, EV-D68 is propagated well at 35 °C.
  5. The time of incubation of the MTS dilution depends on the cell type and cell density. The cells should stay in the incubator until a brown color appears in the CC wells (B11-G11) after the MTS solution has been added. The OD value of the CC should be between 0.6 and 1 to stay in the linear range. The incubation time is approximately 40 to 60 min for Hela Rh cells at 37 °C.
  6. The absorbance value of brown-colored formazan product can reach the biggest at wavelength of 498 nm.
  7. In general, this assay/method is adaptable for other picornaviruses with a few considerations (e.g., cell types, medium and temperature).


  1. Growth culture medium
    MEM supplemented with:
    10% fetal calf serum
    2 mM L-glutamine
    0.075% sodium bicarbonate
  2. Assay medium
    MEM supplemented with:
    2% fetal calf serum
    2 mM L-glutamine
    0.075% sodium bicarbonate
    30 mM MgCl2
  3. MTS/PMS solution
    1. 1 g of MTS powder is dissolved in 500 ml of PBS and stirred
    2. Adjust pH of solution with 1 N HCl to be between 6.0-6.5
    3. 23 mg of PMS powder is added until complete dissolution by stirring
    4. Filter the solution through a rapid-flow sterile disposable filter unit
    5. 3 ml aliquot solution is stored at -20 °C (it should be kept in the dark to avoid degradation)


This work was supported by a fellowship to Liang Sun from China Scholarship Council (CSC) (grant 201403250056) and the Interuniversitaire attractiepolen (IUAP) BELVIR. The protocol described here was based on the following paper: Sun et al. (2015).


  1. Lacroix, C., Querol-Audi, J., Roche, M., Franco, D., Froeyen, M., Guerra, P., Terme, T., Vanelle, P., Verdaguer, N., Neyts, J. and Leyssen, P. (2014). A novel benzonitrile analogue inhibits rhinovirus replication. J Antimicrob Chemother 69(10): 2723-2732.
  2. Sun, L., Meijer, A., Froeyen, M., Zhang, L., Thibaut, H. J., Baggen, J., George, S., Vernachio, J., van Kuppeveld, F. J., Leyssen, P., Hilgenfeld, R., Neyts, J. and Delang, L. (2015). Antiviral activity of broad-spectrum and enterovirus-specific inhibitors against clinical isolates of enterovirus D68. Antimicrob Agents Chemother 59(12): 7782-7785.



背景 作为新出现的病原体,以前很少报道靶向EV-D68的抗病毒化合物。迫切需要建立和开发抗病毒方法来对抗潜在的EV-D68流行病。在这里,我们报告一个详细的方案,可用于识别选择性抗EV-D68化合物。为了筛选和鉴定潜在的抗病毒化合物,基于MTS的CPE降解测定法是可重复使用的,易于使用和省时的方法,被广泛使用。该方法依赖于化合物抑制EV-D68诱导的CPE降低的能力。当化合物主动抑制EV-D68的复制时,病毒诱导的CPE将被减少或不存在。因此,代谢活性细胞能够将黄色四唑盐底物(MTS / PMS)转化为棕色甲an制品。当化合物不具有抗病毒作用时,宿主细胞死于病毒诱导的CPE,其将缺乏代谢活性,并且黄色底物保持未代谢。比色转化是定量的,因为可以从该测定产生的数据计算EC 50。

关键字:肠道病毒D68, 抗病毒测定, CPE, MTS/PMS溶液, 细胞培养


  1. 离心管14ml(Corning,Falcon ®,目录号:352059)
  2. 1.5 ml Eppendorf Snap-Cap微量离心管(VWR,目录号:21008-959)
  3. 4毫升螺旋颈瓶,琥珀色玻璃(VWR,目录号:548-0052)
  4. 透明的96孔(平底)组织培养板(Corning,Falcon ,目录号:353072)
  5. 组织培养瓶,150cm 2(TPP,目录号:90856)
  6. 移液头(10μl,100μl,1,000μl)
  7. 一次性血清移液管(5ml,10ml和25ml)
  8. 试剂储存器,25 ml(Biotix,目录号:SR-0025-5SWM)
  9. 具有SFCA膜的Nalgene快速无菌一次性过滤器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:156-4020)
  10. Hela Rh细胞系(Hela亚克隆,高度易感和允许使用鼻病毒诱导的CPE,来自比利时的Janssen Pharmaceutica的礼物)(Lacroix等人,2014年)
  11. 所有EV-68菌株均在HeLa Rh细胞上培养,储存于-80°C
    1. 从荷兰分离出EV-D68菌株(742,947,2042,1348,2284和670)(Adam Meijer的一种礼物,国家公共卫生和环境研究所的诊断与筛选[RIVM],Bilthoven) />
    2. EV-D68菌株(US/KY/14-18953,US/IL/14-18952和US/MO14-18947) - 这些菌株最初获自BEI Resources
  12. Dulbecco的磷酸盐缓冲盐水(DPBS),不含钙,无镁(Thermo Fisher Scientific,Gibco TM,目录号:14190144)
  13. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)
  14. 最低必需培养基(MEM)Rega-3(Thermo Fisher Scientific,Gibco TM ,目录号:19993013)
  15. 潜在的抗病毒化合物
    1. SG85 *
    2. Pleconaril *
    3. Vapendavir *
    4. 皮罗达维尔*
    5. Rupintrivir *
    6. Enviroxime *
    7. 法维西亚*
  16. 胎牛血清(GE Healthcare,Hyclone TM,目录号:SH30084.03)
  17. L-谷氨酰胺200mM(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  18. 碳酸氢钠7.5%溶液(Thermo Fisher Scientific,Gibco TM,目录号:25080060)
  19. 最低必需培养基(MEM),无谷氨酰胺,无酚红(Thermo Fisher Scientific,Gibco TM,目录号:51200038)
  20. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  21. CellTiter 96 AQueous MTS试剂粉末(Promega,目录号:G1111)
  22. HCl
  23. 二甲基亚砜(VWR,目录号:67-68-5)
  24. 生长培养基(见食谱)
  25. 测定培养基(参见食谱)
  26. MTS/PMS解决方案(见配方)


  1. 移液器控制器,PIPETBOY acu2(VWR,目录号:612-0927)
  2. 多通道移液管,10-100μl(Eppendorf)
  3. 37℃(和35℃)和5%CO 2(粘结剂)的培养箱
  4. 中型台式离心机(Sigma Laborzentrifugen,模式:4K15C)
  5. Moxi TM Z迷你自动细胞计数器(ORFLO Technologies,目录号:MXZ001)
  6. SpectraMax 190酶标仪(Molecular Devices,型号:SpectraMax 190)
  7. pH仪表(Xylem Analytics,WTW,型号:inoLab 9310 IDS)
  8. 倒置显微镜(Motic,型号:AE21)
  9. 安全柜(Telstar)


  1. GraphPad Prism


  1. 在37℃和5%CO 2下,生长Hela Rh细胞在生长培养基(见食谱)中汇合。
    注意:Hela Rh细胞在10分钟内传播1次,3-4天后会汇合。
  2. 吸出上清液,用10ml PBS洗涤细胞三次,用37℃的胰蛋白酶将细胞分离3-5分钟。
  3. 当细胞分离时,用10ml生长培养基在14ml管中收集细胞。在室温下以269×g离心细胞6分钟。
  4. 将细胞沉淀重新悬浮在10ml测定培养基中(参见食谱)。
  5. 计数单元格数。将细胞以每孔100μl测定培养基以15,000个细胞的密度将细胞种植到96孔板中。在培养箱中37℃和5%CO 2孵育细胞24小时,以使细胞粘附在板上。
  6. 将化合物稀释至测定培养基中所需的起始浓度。该起始浓度应为所需的最终浓度的6倍以准备1对3连续稀释。在第二列中加入50μl的6x化合物稀释液至100μl测定培养基中,加入接种细胞的孔B2至G2(最多6个化合物,图1)。

    图1.用于抗病毒测定的96孔板的布局。灰井仅含有培养基; VC:病毒控制,CC:细胞控制。

  7. 通过温和移液混合第2列培养基,并将50μl培养基从该柱转移到下一列3(B3-G3)(参见图2A);重复第9列(B9-G9)。从第9列弃去50μl培养基。第10列(B10-G10)将是病毒对照(VC,测定培养基+ EV68),第11列(B11-G11)将是细胞对照(CC,仅测定培养基)。
  8. 将100μl所需稀释度的EV68(MOI = 0.001)添加到第2-10列和100μl测定培养基至第11列(参见图2B)。 96孔板边缘的孔(见图1,标记为灰色)含有200μl培养基,但不会使用,因为边缘效应可能会影响测定结果。

    图2.工作流程。 A.在处理和感染的孔中轻轻混合测定培养基。将50μl培养基转移到下一个孔,以制备1-in-3稀释系列。 B.制备复合稀释液后,加入100μl含有2x病毒稀释液的测定培养基。

  9. 将96孔板置于培养箱(35℃,5%CO2和95%相对湿度)中3-4天,直到通过VC孔中的显微镜观察到完全病毒诱导的细胞病变效应(CPE)(见图3 )。

    图3.细胞对照孔(A)和病毒对照孔(B)中的HeLa Rh细胞。 比例尺= 35μm。

  10. 取出第2-11列中所有孔的测定培养基,并加入100μl5%MTS/PMS稀释于酚红和无谷氨酰胺培养基中。
  11. 在37℃5%CO 2培养箱中孵育40-60分钟后,用波长498nm的酶标仪测量96孔板的每个孔中的吸光度。 />


  1. 抗病毒活性计算如下:
    %抗病毒活性=(OD EV68 +化合物 VC )/(OD CC -
    OD< CC>和OD< VC>分别表示细胞对照(第11列)和病毒对照(第10栏)的光密度(OD)的平均值, > OD EV68 +化合物对应于每个感染和处理良好的OD值
  2. 计算每种化合物的EC 50值,其定义为病毒诱导的CPE降低50%的化合物浓度。通过GraphPad Prism生成的曲线拟合来计算抗病毒活性的这个值 注意:或者,如果GraphPad Prism不可用,也可以使用对数插值来估计EC 50值。下面的代表数据由对数插值显示。


  1. %计算每种浓度的抗病毒活性,如下所示:

    EC 50 = 10 ((Log( A ) - Log( B ))x(( C - 50)/( C -   D ))+日志( B )) /> 哪里,
    A :%抗病毒活性的化合物浓度< 50%
    B :%抗病毒活性的化合物浓度> 50%
    C :%antiviral activity的值> 50%
    D :%antiviral activity< 50%
  2. 蛋白酶抑制剂SG85对不同EV68临床分离株的抗病毒活性的剂量 - 反应曲线的实例(参见图4)。 EC50值出现在Sun等人的中。 (2015)



  1. 根据抗病毒测定中使用的细胞类型,每孔的细胞数量可能需要优化。细胞对照组中的细胞在3或4天后不会过度生长,这一点很重要
  2. 使用最低的病毒接种,在感染后第3天或第4天在所有孔中仍然产生100%的完全CPE。
  3. 如果化合物的1 - 2连续稀释是优选的,则制备4x浓缩的化合物稀释液,并将100μl该化合物稀释液加入第2列;然后从井中转移100μl,以产生1:2稀释系列。
  4. 由于其最佳生长温度低,所以EV-D68在35℃下很好地传播
  5. MTS稀释液的孵育时间取决于细胞类型和细胞密度。在添加MTS溶液后,细胞应留在培养箱中,直到CC孔(B11-G11)中出现褐色。 CC的OD值应在0.6和1之间,保持在线性范围内。 Hela Rh细胞在37℃下的孵育时间约为40-60分钟
  6. 棕色甲an制品的吸光度值在波长498 nm处可达到最大值。
  7. 一般来说,该测定/方法适用于其他微小核糖核酸病毒,具有少量考虑(例如,细胞类型,培养基和温度)。


  1. 生长培养基
    2mM L-谷氨酰胺
  2. 测定培养基
    2mM L-谷氨酰胺
    30mM MgCl 2
  3. MTS/PMS解决方案
    1. 将1g MTS粉末溶解于500ml PBS中并搅拌
    2. 用1N HCl调节溶液的pH值在6.0-6.5之间
    3. 加入23mg PMS粉末直至通过搅拌完全溶解
    4. 通过快速流动的无菌一次性过滤器单元过滤溶液
    5. 将3ml等分溶液储存在-20℃(应保持在黑暗中以避免降解)


这项工作得到了中国奖学金委员会(CSC)(授权201403250056)和跨国公司(IUAP)BELVIR的梁孙奖学金的支持。这里描述的协议是基于以下论文:Sun等人。 (2015)。


  1. Lacroix,C.,Querol-Audi,J.,Roche,M.,Franco,D.,Froeyen,M.,Guerra,P.,Terme,T.,Vanelle,P.,Verdaguer,N.,Neyts,J 。和Leyssen,P.(2014)。一本小说苄腈类似物抑制鼻病毒复制。 J Antimicrob Chemother 69(10):2723-2732。
  2. Sun,L.,Meijer,A.,Froeyen,M.,Zhang,L.,Thibaut,HJ,Baggen,J.,George,S.,Vernachio,J.,van Kuppeveld,FJ,Leyssen,P.,Hilgenfeld ,R.,Neyts,J.and Delang,L。(2015)。针对肠道病毒D68临床分离株的广谱和肠道病毒特异性抑制剂的抗病毒活性。抗微生物剂Chemother 59(12):7782-7785。
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引用:Sun, L., Delang, L., Mirabelli, C. and Neyts, J. (2017). In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68. Bio-protocol 7(6): e2183. DOI: 10.21769/BioProtoc.2183.