Neutral Red Assay for Murine Norovirus Replication and Detection in a Mouse

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PLOS Pathogens
Jul 2012



Neutral red (NR) is a dye that must be actively imported into the cell, and, therefore, the dye has been used for decades to selectively stain living cells. In addition, NR can also be incorporated into virus particles, although the mechanism behind this is poorly understood. Once encapsulated into the virion, NR, a light sensitive dye, can be photoactivated to inactivate the virus. The proposed mechanism explaining this observation is that activation of NR allows the dye to cross-link viral genome to viral capsid and thus preventing viral uncoating and infection. To study the early events of murine norovirus (MNV)-host interaction, light-sensitive NR-containing MNV is used to distinguish between input virus (i.e., NR-containing virus) and replicated virus (i.e., NR-free virus). This protocol describes the incorporation of NR into MNV capsids and the use of these virions for detection of viral replication in a mouse and in tissue culture by standard plaque assay. The same technique is also used for study of poliovirus replication (1-3). Thus, there is the potential that this technique can be used for additional non-enveloped viruses. However, this has to be tested on a case-by-case basis as unpublished data on feline calicivirus suggests not all viruses may be able to stably incorporate NR into their capsid (J. Parker, personal communication).

Materials and Reagents

  1. Neutral red 0.33% solution (Sigma-Aldrich, catalog number: N2889 )
  2. DMEM/ High glucose (Hyclone, catalog number: SH30243.02 )
  3. 2x MEM (Life Technologies, Gibco®, catalog number: 11935 )
  4. 100x Penicillin and streptomycin (Hyclone, catalog number: SV30010 )
  5. 100x Non-essential amino acids (Hyclone, catalog number: SH30238.01 )
  6. 1 M HEPES (Hyclone, catalog number: SH30237.01 )
  7. 200 mM (100x) L-glutamine (Hyclone, catalog number: SH30034.01 )
  8. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10437 or Hyclone, catalog number: SH30070.02 )
  9. RAW 264.7 cell line (ATCC, catalog number: TIB-71 )
  10. Murine norovirus (MNV-1; GV/MNV1/2002/USA)
  11. Sea Plaque Agarose (Lonza, catalog number: 50100 )
  12. Aluminum foil
  13. 1.0 mm Zirconia/Silica beads (BioSpec Products, catalog number: 11079110z )
  14. Mice (6-8 weeks old Balb/cJ)
  15. DMEM-10 (see Recipes)
  16. DMEM-5 (see Recipes)
  17. 2x MEM media (500 ml) (see Recipes)
  18. SeaPlaque agarose (see Recipes)
  19. 1:1 ratio of molten media (see Recipes)


  1. 175 cm2 flask (Corning, catalog number: 3292 )
  2. Model 35 Speed Rocker (Labnet, catalog number: S2035 )
  3. Tissue culture incubator (Sanyo, catalog number: MCO-36M )
  4. Magna Lyser Bead beater (F. Hoffmann-La Roche, catalog number: 0 3358968001 )
  5. Photographic safe red light


  1. Neutral red virus preparation
    1. Seed RAW 264.7 cells in a total of 30 ml of DMEM-10 media at a density of 4 x 107 cells in a 175 cm2 flask and incubate in a tissue culture incubator at 37 °C and 5% CO2 overnight.
    2. To obtain a virus stock, infect cells the next day with MNV by adding MNV at a multiplicity of infection (MOI) of 0.05 and incubate for 1 h in a tissue culture incubator at 37 °C and 5% CO2.
    3. After the hour, add neutral red at 0.001% v/v from a 0.33% stock solution to the cells.
    4. Wrap the entire flask with aluminum foil including the filter cap of the flask.
    5. Incubate in a tissue culture incubator at 37 °C and 5% CO2 for 48 h.
    6. After the 48 h, generate a freeze/thaw lysate. Freeze cells by moving the entire flask (including cells and media) covered with aluminum foil to -80 °C for at least 60 min. Then thaw flask contents at room temperature or in a tissue culture incubator at 37 °C until all ice has melted. Repeat freeze/thaw a second time. This will be your virus stock.
      Note: After the second freeze/thaw cycle, virus stock should be aliquoted (in a darkened room) into single-use aliquots (e.g., 1 ml) and stored at -80 °C since light-sensitivity of virus stock decreases after repeated freeze/thaw cycles.
    7. Block out all light in your tissue culture room to make a dark room and equip with a safe red light used in photography dark rooms.
    8. Perform plaque assay to determine viral titer of your virus stock as previously described in detail (2) and as described briefly below. The day before the plaque assay seed 2 x 106 Raw 264.7 cells in DMEM-10/ well in six well plates.
    9. On the day of the plaque assay, perform 10-fold serial dilutions of your virus stock in DMEM-5. Make sure to adjust the volume for duplicate plaque assays, i.e., you will need a total volume of 2.5 ml for each dilution.
    10. Next, take the six well plates with cells seeded on the day before, aspirate the media and infect cells in the dark in duplicate wells for each dilution with 0.5 ml/ well, wrap in aluminum foil and rock for 1 h at room temperature with a speed rocker at ~18 oscillations per min.
      Note: The 1 h incubation was determined experimentally to ensure efficient entry and uncoating of MNV. However, the length of time necessary for other viruses may vary.
    11. Turn on the lights and leave the remaining 10-fold viral dilutions in the light for at least 10 min.
    12. Repeat step 1-j. but keep plates exposed to light (i.e., do not wrap plates in aluminum foil).
    13. After the hour, finish the plaque assay for both sets of plates with or without aluminum foil in the light by adding 2 ml/well of a 1:1 ratio of molten media (1:1 ratio of SeaPlaque agarose and 2x MEM). To allow agarose to solidify incubate plates at room temperature for 10 min. Then, move plates to a tissue culture incubator and incubate at 37 °C, 5% CO2 for 48 h.
    14. After 48 h stain cells with a neutral red solution overlay (0.01% neutral red in 1x PBS), for 1-3 h prior to aspirating the solution and counting plaques as described previously in detail (Gonzalez-Hernandez et al., 2012). 
      Note: To calculate viral titers and obtain plaque forming units per milliliter (PFU/ml), add the number of plaques in both wells at a single dilution and multiply by the dilution factor (i.e., 1 ml if 2 wells are infected with 0.5 ml). Figure 1 shows an example of a 6 well plate with plaques. To calculate the viral titer in the example: 11 + 9 = 20 plaques; 20 x 102 dilution factor = 2 x 103 PFU/ml. Two- to three-log reductions are typically observed in viral titers comparing total virus titers (i.e. obtained in dark) and light-insensitive (i.e. obtained in light) infections. Depending on the viral strain, log reductions may differ.
  2. Tissue culture infection with neutral red virus
    1. Seed 2 x 106 Raw 264.7 cells in six well plates the day before.
    2. The next day, block out all light in your tissue culture room to make a dark room and equip with a safe red light used in photography dark rooms.
    3. Infect with MNV containing NR at an MOI of 0.001 for 1 h.
      Note: Pre-treatments with inhibitors of viral entry or uncoating can be performed before incubating with virus. Post-treatments with the same inhibitors are performed as a control after the 1 h incubation with virus as described (Perry, 2010).
    4. Flash with light for various lengths of times between 0 to 60 min at the end of the infection time.
      Note: For MNV, times between 0 and 60 min are typically used and at least 10 min has been sufficient to inactivate any leftover input virus (Perry, 2010).
    5. Overlay with 2 ml of a 1:1 ratio of molten media (1:1 ratio of SeaPlaque agarose and 2x MEM), as you would do for plaque assays and as described  (Gonzalez-Hernandez et al., 2012; Perry et al., 2012).
    6. Incubate for 48-72 h in a tissue culture incubator at 37 °C and 5% CO2.
    7. Count plaques by adding the number of plaques in both wells at a single dilution and multiplying by the dilution factor, as described above and previously(Gonzalez-Hernandez et al., 2012; Perry et al., 2012).
  3. Mouse infection with neutral red virus
    1. Inoculate mice perorally by pipetting virus directly into the mouth or by oral gavage with NR-MNV in a darkened room with a safe red light.
      Note: We infected six- to eight-week old Balb/c mice perorally with NR-MNV at 105 pfu. Depending on the experiment, the length of infection will vary. We typically infect mice for 12 –72 h. At the experimental endpoint (i.e. 12 h post-infection), sacrifice mice in a darkened room with a safe red light and harvest different regions of the gastrointestinal tract, each 1 cm in lengths. Place each tissue piece in an individual 2 ml centrifuge screw cap tube containing 0.4 - 0.5 g of silica beads and 1 ml of DMEM-10.
    2. Homogenize tissues in the dark with a bead beater for 60 min at 6,000 rpm, as described (Gonzalez-Hernandez et al., 2012).
    3. Freeze (at -80 °C) and thaw tissues (at 37 °C) once before performing parallel plaque assays in the dark and following light exposure as described above.
    4. Count plaques 48 h later.
      Note: MNV-1 is known to undergo the first round of replication in ~12 h. Therefore, samples collected 3 h post-infection should only show input virus (i.e., virus containing NR and sensitive to light).

Figure 1. Representative image of a plaque assay plate after 48 h of MNV-1 infection. Plaques are observed after staining with a neutral red solution overlay for 1-3 h. The image shows duplicate wells of three 10-fold viral dilutions: 10-1, 10-2 and 10-3 dilutions. Arrows indicate the formation of plaques and the viral titer of the sample is indicated below. Answer: 11+9= 20 x 102 = 2 x 103 PFU/ml.


  1. DMEM-10
    DMEM/High glucose (1 L)
    10% low-endotoxin FBS
    1% HEPES
    1% Penicillin and streptomycin
    1% Non-essential amino acids
    1% L-glutamine
  2. DMEM-5
    DMEM/High glucose (1 L)
    5% low-endotoxin FBS
    1% HEPES
    1% Penicillin and streptomycin
    1% Non-essential amino acids
    1% L-glutamine
  3. 2x MEM media (500 ml)
    10% low-endotoxin FBS
    1% HEPES
    1% Penicillin and streptomycin
    2% L-glutamine
    Equilibrate to 37 °C
  4. SeaPlaque agarose
    1.5 g SeaPlaque agarose
    50 ml water
    Autoclave solution
    Equilibrate to 42 °C
  5. 1:1 ratio of molten media
    Mix 50 ml of 2x MEM and 50 ml of SeaPlaque agarose a 1:1 before adding to the cells


This neutral red assay protocol for murine norovirus was adapted thanks to the original work for poliovirus that was previously described by Brandenburg et al. (2007) and Kuss et al. (2008). Additionally, this protocol was adapted from the work performed by Perry et al. (2010); Perry et al. (2012) and Gonzalez-Hernandez et al. (2012). Our work was funded by start-up funds from the University of Michigan and NIH grant AI080611 to C.E.W. M.B.G.-H. was funded by the University of Michigan Experimental Immunology Training Grant (NIH T32 A1007413-16), by the Molecular Mechanisms of Microbial Pathogenesis Training Grant (NIH T32 A1007528), and by the Herman and Dorothy Miller Fund for Innovative Immunology Research. J.W.P. was funded by the University of Michigan Human Genetics training grant (grant NIH T32 GM 07544) and the Molecular Mechanisms of Microbial Pathogenesis training grant (NIH T32 AI 007528).


  1. Brandenburg, B., L. Y. Lee, M. Lakadamyali, M. J. Rust, X. Zhuang, and J. M. Hogle. 2007. Imaging Poliovirus entry in live cells. PLoS Biol 5(7): e183.
  2. Gonzalez-Hernandez, M. B., J. Bragazzi Cunha, and C. E. Wobus. 2012. Plaque assay for murine norovirus. J Vis Exp (66): e4297.
  3. Kuss, S. K., C. A. Etheredge, and J. K. Pfeiffer. 2008. Multiple host barriers restrict Poliovirus tTrafficking in mice. PLoS Pathog 4(6): e1000082.
  4. Perry, J. W., M. Ahmed, K.-O. Chang, N. J. Donato, H. D. Showalter, and C. E. Wobus. 2012. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response. PLoS Pathog 8(7): e1002783.
  5. Perry, J. W., and C. E. Wobus. 2010. Endocytosis of murine norovirus 1 into murine macrophages is dependent on dynamin II and cholesterol. J Virol  84(12): 6163-6176.


中性红(NR)是必须主动导入细胞的染料,因此,染料已经使用数十年来选择性地染色活细胞。此外,NR也可以被并入病毒颗粒中,虽然其背后的机制知之甚少。一旦包封入病毒粒子中,NR(一种光敏染料)可以被光活化以使病毒失活。解释这种观察的提出的机制是NR的活化允许染料将病毒基因组交联病毒衣壳,从而防止病毒脱壳和感染。为了研究鼠诺如病毒(MNV) - 宿主相互作用的早期事件,使用光敏感的含NR的MNV来区分输入病毒(即含有NR的病毒)和复制的病毒( >即,无NR的病毒)。该方案描述了将NR掺入MNV衣壳中以及这些病毒颗粒用于通过标准噬菌斑测定在小鼠和组织培养物中检测病毒复制的用途。相同的技术也用于研究脊髓灰质炎病毒复制(1-3)。因此,存在该技术可用于另外的非包膜病毒的潜力。然而,这必须在逐案基础上进行测试,因为关于猫杯状病毒的未公开数据表明不是所有的病毒都能够将NR稳定地掺入其衣壳中(J.Parker,personal communication)。


  1. 中性红0.33%溶液(Sigma-Aldrich,目录号:N2889)
  2. DMEM /高葡萄糖(Hyclone,目录号:SH30243.02)
  3. 2x MEM(Life Technologies,Gibco ,目录号:11935)
  4. 100x青霉素和链霉素(Hyclone,目录号:SV30010)
  5. 100x非必需氨基酸(Hyclone,目录号:SH30238.01)
  6. 1 H HEPES(Hyclone,目录号:SH30237.01)
  7. 200mM(100x)L-谷氨酰胺(Hyclone,目录号:SH30034.01)
  8. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10437或Hyclone,目录号:SH30070.02)
  9. RAW 264.7细胞系(ATCC,目录号:TIB-71)
  10. 鼠诺如病毒(MNV-1; GV/MNV1/2002/USA)
  11. 海斑琼脂糖(Lonza,目录号:50100)
  12. 铝箔
  13. 1.0mm氧化锆/二氧化硅珠(BioSpec Products,目录号:11079110z)
  14. 小鼠(6-8周龄Balb/cJ)
  15. DMEM-10(参见配方)
  16. DMEM-5(见配方)
  17. 2x MEM培养基(500ml)(参见配方)
  18. SeaPlaque琼脂糖(参见食谱)
  19. 1:1的熔融介质比率(见配方)


  1. 175cm 2烧瓶(Corning,目录号:3292)
  2. Model 35 Speed Rocker(Labnet,目录号:S2035)
  3. 组织培养箱(Sanyo,目录号:MCO-36M)
  4. Magna Lyser Bead Beater(F.Hoffmann-La Roche,目录号:03358968001)
  5. 摄影安全红光


  1. 中性红色病毒制备
    1. 种子RAW 264.7细胞在175cm 2烧瓶中的总共30ml的DMEM-10培养基中以4×10 7个细胞的密度接种,并在组织培养物 培养箱中37℃和5%CO 2过夜
    2. 为了获得病毒母液,通过以0.05的感染复数(MOI)加入MNV并在37℃和5%CO 2下在组织培养箱中温育1小时,用MNV感染细胞, sub>。
    3. 一小时后,将0.001%v/v的中性红从0.33%储备溶液中加入到细胞中
    4. 用包括烧瓶过滤盖的铝箔包裹整个烧瓶
    5. 在组织培养孵育器中在37℃和5%CO 2孵育48小时。
    6. 48小时后,产生冷冻/融化裂解物。通过将用铝箔覆盖的整个烧瓶(包括细胞和培养基)移动至-80℃至少60分钟来冷冻细胞。然后在室温下或在37℃的组织培养箱中解冻烧瓶内容物,直到所有的冰融化。重复冻结/解冻第二次。这将是您的病毒储备。
    7. 阻挡组织培养室中的所有光线,使其成为暗室,并配备用于摄影黑暗房间的安全红光。
    8. 如先前详细描述的(2)和如下简述,进行空斑测定以确定病毒储液的病毒滴度。斑块测定前一天在DMEM-10 /孔中在6孔板中接种2×10 6个Raw 264.7细胞。
    9. 在噬斑测定的当天,在DMEM-5中进行10倍连续稀释的病毒原种。请务必调整重复空斑测定的体积,即, ,每次稀释的总体积为2.5ml。
    10. 接下来,取出具有在前一天接种的细胞的六孔板,抽吸培养基并在黑暗中以0.5ml /孔每孔稀释一式两份感染细胞,在室温下用铝箔和岩石包裹1小时,高速摇臂每分钟〜18次振动。
    11. 打开灯,将剩余的10倍病毒稀释液放在灯中至少10分钟。
    12. 重复步骤1-j。但保持板暴露于光(,即,不要在铝箔包装板)。
    13. 1小时后,通过加入2ml /孔的1:1比率的熔融培养基(1:1比例的SeaPlaque琼脂糖和2×MEM),在光照下通过或不使用铝箔,完成两组板的空斑测定。为了允许琼脂糖凝固,将板在室温下孵育10分钟。然后,将板移动到组织培养孵育器并在37℃,5%CO 2孵育48小时。
    14. 在48小时后,在吸出溶液并计数噬菌斑之前用中性红色溶液覆盖(在1×PBS中的0.01%中性红)染色细胞1-3小时,如Gonzalez-Hernandez等人,/em>,2012)。
      注意:为了计算病毒效价并获得每毫升噬菌斑形成单位(PFU/ml),以单一稀释度在两个孔中加上噬菌斑数,并乘以稀释因子(即,如果2个孔被感染,则为1ml)与0.5ml)。图1显示了具有斑块的6孔板的实例。为了计算实施例中的病毒滴度:11 + 9 = 20个噬菌斑; 20 x 10 2 ml PFU/ml。通常在比较总病毒滴度(即在黑暗中获得)和光不敏感(即在光照下获得)感染中在病毒效价中观察到2至3个对数级的减少。根据病毒株,对数减少可能不同。
  2. 组织培养物感染与中性红色病毒
    1. 种子2×10 6个原始264.7细胞在前一天的六孔板中
    2. 第二天,阻止你的组织培养室里的所有光,使一个黑暗的房间,并配备用于摄影黑暗房间的安全红灯。
    3. 用含有NR的MNV以0.001的MOI感染1小时 注意:在与病毒孵育之前可以进行使用病毒进入或脱壳抑制剂的预处理。在与病毒孵育1小时后,用相同的抑制剂进行后处理(Perry,2010 )。
    4. 闪光灯在感染时间结束时在0至60分钟之间的不同长度的光 注意:对于MNV,通常使用0至60分钟的时间,并且至少10分钟足以灭活任何剩余输入病毒(Perry,2010)。
    5. 用2ml 1:1比率的熔融培养基(1:1比率的SeaPlaque琼脂糖和2×MEM)覆盖,如同对于噬菌斑测定和如Gonzalez-Hernandez等人 >,2012; Perry et al。,2012)。
    6. 在组织培养箱中在37℃和5%CO 2下孵育48-72小时。
    7. 如上所述和先前所述,通过将单孔稀释中的噬斑数目乘以稀释倍数来计数噬菌斑(Gonzalez-Hernandez等人,2012; Perry等人。, 2012)。
  3. 鼠标感染与中性红色病毒
    1. 通过将病毒直接吸入口中或通过用安全红灯在黑暗的房间中用NR-MNV口服管饲来口服小鼠。

      pfu下口感六到八周龄的Balb/c小鼠。根据实验,感染的长度将不同。我们通常感染小鼠12-72小时。在实验终点(即感染后12小时),在暗室中用安全的红光处死小鼠,并收获胃肠道的不同区域,每个长度为1cm。将每个组织片放在含有0.4-0.5g二氧化硅珠和1ml DMEM-10的单独的2ml离心螺旋盖管中。
    2. 按照Gongzalez-Hernandez等人,2012年所述,使用珠磨机在6,000rpm下匀浆组织在黑暗中60分钟。
    3. 在黑暗中和在如上所述的光照下进行平行空斑测定之前,冷冻(在-80℃)和融化组织(在37℃)一次。
    4. 48小时后计数斑块。

图1.在MNV-1感染48小时后噬菌斑测定板的代表性图像。在用中性红色溶液覆盖染色1-3小时后观察噬斑。该图显示了三个10倍病毒稀释液的两个孔:10个-1,10个,10个,10个和10个稀释。箭头表示噬菌斑的形成,样品的病毒滴度如下所示。答案:11 + 9 = 20 x 10 2 = 2 x 10 3 PFU/ml。


  1. DMEM-10
    DMEM /高葡萄糖(1L)
    1%青霉素和链霉素 1%非必需氨基酸
  2. DMEM-5
    DMEM /高葡萄糖(1L)
    5%低内毒素FBS 1%HEPES
    1%青霉素和链霉素 1%非必需氨基酸
  3. 2×MEM培养基(500ml) 10%低内毒素FBS
    1%青霉素和链霉素 2%L-谷氨酰胺 平衡至37°C
  4. SeaPlaque琼脂糖
    1.5克SeaPlaque琼脂糖 50ml水
  5. 1:1的熔融介质比率 在加入细胞
    之前,将1:1的50ml 2×MEM和50ml的SeaPlaque琼脂糖混合


这种用于鼠诺如病毒的中性红测定方案由于先前由Brandenburg等人(2007)和Kuss等人(2008)描述的脊髓灰质炎病毒的原始工作而改编, )。此外,该方案改编自Perry等人(2010)所做的工作; Perry等人(2012)和Gonzalez-Hernandez等人(2012)。我们的工作由密歇根大学和NIH授权AI080611至C.E.W.的启动资金资助。 M.B.G.-H.由密歇根大学实验免疫学培训授权(NIH T32 A1007413-16),微生物病原学培养授权分子机制(NIH T32 A1007528)和Herman和Dorothy Miller基金创新免疫学研究资助。 J.W.P.由密歇根大学人类遗传学培训授予(授予NIH T32 GM 07544)和微生物发病机理分子机制训练授权(NIH T32 AI 007528)资助。


  1. Brandenburg,B.,L.Y.Lee,M.Lakadamyali,M.J.Rust,X.Bhuang和J.M.Hogle。 2007. 成像

    P 条目。 PLoS Biol 5(7):e183

  2. Gonzalez-Hernandez,M.B.,J.Bragagi Cunha和C.E.Wobus。 2012. 鼠诺如病毒的噬斑测定法。 J Vis Exp (66):e4297。
  3. Kuss,S.K.,C.A.Etheredge,和J.K.Pfeiffer。 2008. 多台主机障碍限制了脊髓灰质炎病毒 tTraffic in mice 。 PLoS Pathog 4(6):e1000082。
  4. Perry,J.W.,M.Ahmed,K.-O. Chang,N.J.Donato,H.D. Showalter和C.E.Wobus。 2012. 小分子去泛素酶抑制剂的抗病毒活性通过诱导解折叠蛋白反应发生。 PLoS Pathog 8(7):e1002783。
  5. Perry,J.W。,和C.E.Wobus。 2010. 将鼠诺罗病毒1内吞到鼠巨噬细胞中依赖于dynamin II和胆固醇。 J Virol  84(12):6163-61 76。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. González-Hernández, M. B., Perry, J. W. and Wobus, C. E. (2013). Neutral Red Assay for Murine Norovirus Replication and Detection in a Mouse. Bio-protocol 3(7): e415. DOI: 10.21769/BioProtoc.415.
  2. Perry, J. W., M. Ahmed, K.-O. Chang, N. J. Donato, H. D. Showalter, and C. E. Wobus. 2012. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response. PLoS Pathog 8(7): e1002783.