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Virus Infection and Titration of SARS-CoV in Mouse Lung
小鼠肺部非典型性肺炎病毒的感染和滴定测试

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Abstract

Two critical steps when investigating an animal model of a virus infection are consistently successfully infecting animals and accurately determining viral titers in tissue throughout the course of infection. Here we discuss in detail how to infect mice with SARS-CoV and then quantify the titer of virus in the lung.

Keywords: SARS-CoV(SARS-CoV), Virus titration(病毒滴定), Virus infection(病毒感染), Lung preparation(肺的制备)

Materials and Reagents

  1. Mice (NCI) (Balb/c or C57BL/6, 4 weeks to 18+ months)
  2. Vero E6 cells (ATCC, catalog number: CRL-1586 )
  3. Isofluorane (USP inhalation vapour; liquid) ( NDC: 57319-559-06 )
  4. Dulbecco’s modified eagle medium high glucose (DMEM) (Life Technologies, Gibco®, catalog number: 11965092 )
  5. 100 mg/ml ketaset III ketamine HCl injection (USP) (DEA Schedule II Drug) (NDC: 0856-2013-01 )
  6. 100 mg/ml AnaSed injection xylazine (Lloyd Laboratories, NADA number: 139-236 )
  7. 0.9% sodium chloride irrigation (USP) (Baxter, catalog number: 2F7124 )
  8. 1x Dulbecco’s phosphate buffered saline (DPBS) (Life Technologies, Gibco®, catalog number: 14190-144 )
  9. Formaldehyde solution (Sigma-Aldrich, catalog number: 252549 ) (37 wt.% in H2O)
  10. Crystal violet (Sigma-Aldrich, catalog number: C0775 )
  11. 25 mM DMEM with hepes (Life Technologies, Gibco®, catalog number: 32430 )
  12. 200 mM L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  13. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150 )
  14. MEM non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050 )
  15. 50 mg/ml gentamicin sulfate (Lonza, catalog number: 17-518Z )
  16. Penicillin streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  17. DMEM (Life Technologies, Gibco®, catalog number: 12100-046 )
  18. Agarose (optimized grade) (Research Products International, catalog number: A20090 )
  19. Ketamine solution (see Recipes)
  20. Culture media (see Recipes)
  21. Overlay media (see Recipes)

Equipment

  1. Biosafety hood in a biosafety level 3 facility
  2. Absorbent pads (Covidien, catalog number: 949)
  3. 2 ml micro tube (Sarstedt AG, catalog number: 72.694.006 )
  4. 5 ml syringe (BD, catalog number: 309646 )
  5. 75 cm2 cell culture flask (canted neck, 0.2 µM vent cap) (Corning, catalog number: 430641 )
  6. 12 well cell culture cluster (flat bottom with lid) (Corning, Costar®, catalog number: 3513 )
  7. 48 well cell culture cluster (flat bottom with lid) (Corning, Costar®, catalog number: 3548 )
  8. 10 ml stripette (Corning, Costar®, catalog number: 3548)
  9. 35 ml closed tissue grinder system (Fisher Scientific, catalog number: 02-542-08 )
  10. Dessicator (Narang Medical, catalog number: P37.1517P )
  11. Gauze Sponges (4 x 4 inch) (Pro Advantage® by NDC, catalog number: P157118 )
  12. 1 ml syringe (BD, catalog number: 309659 )
  13. Precision glide needle (25 G x 5/8) (BD, catalog number: 305122 )
  14. Spray bottle with 70% ethanol
  15. Surgical scissors
  16. Polystyrene foam
  17. Pipetman P200 micropipette
  18. Pipetman P1000 micropipette
  19. CO2 incubator
  20. Pipet aid
  21. Small metal weighing spatula
  22. Tweezers
  23. -80 °C freezer

Procedure

  1. Infection
    1. Arrange materials in a ventilated hood. When using SARS-CoV, all procedures are performed in a biosafety hood in a BSL3 laboratory. When using isofluorane, always anesthetize animals in a fume or biosafety hood.
    2. Thaw virus on ice then dilute in DMEM to the appropriate concentration. Each mouse receives 50 μl of virus.
    3. Place several ply of gauze in the bottom of a plastic dessicator followed by 10 ml of isofluorane.
      Note: Make sure to tape over the center hole in the plastic dessicator grate because small mice are sometimes able to squeeze through this hole and jump into the bottom, isofluorane filled chamber.
    4. Load a P200 micropipette with 50 µl of virus and then secure it in a safe position while placing a mouse on the grate inside the dessicator.
    5. Immediately after the rate of respiration slows, pick up the mouse, hold it vertically and then slowly deliver 50 µl of virus to both nostrils of the mouse. When mice are lightly anesthetized breathing will be deep enough that liquid on the nostrils should be completely aspirated into the lungs.
      Note: The orifice size of the pipetman tips used matters significantly; tips with too small an orifice make it difficult to smoothly dispense virus onto the nostrils.

  2. Removal and processing of lung tissue
    1. A mouse is euthanized by intraperitoneal injection of 300 μl ketamine/xylazine solution. To inject fluid into the intraperitoneal cavity, secure a mouse in the palm of the hand and hold it horizontally. Insert a 25 G x 5/8 g needle at a shallow angle tangential to the mouse and slowly dispense ketamine/xylazine into the peritoneal cavity.
    2. Once the mouse is fully anesthetized, place it on a square of absorbant padding on polystyrene foam and immobilize the mouse by pinning each limb with a 25 G x 5/8 gauge needle.
    3. Use a spray bottle filled with 70% ethanol to sterilize the fur of the mouse.
    4. Use scissors to cut open the abdominal and thoracic cavities by first making an incision from the lower abdominal to the throat of the animal. Opening the abdominal cavity exposes the underside of the diaphragm. Use scissors to pierce through the diaphragm. Cut away the ribcage of the mouse and fully expose the heart and lungs. Cut the heart away from the lungs, and then remove the lungs by cutting the trachea and any remaining connective tissue.
    5. After the lungs are removed from the thoracic cavity, use a 5 ml syringe filled with PBS to wash the blood from the outside of the lungs.
    6. Place the lungs in a tissue grinder and grind them coarsely. Add 3 ml of PBS to the grinder and continue grinding the tissue to a homogenate.
    7. Aliquot 1 ml of homogenate into 2 ml micro tubes, place on dry ice and then transfer to a -80 °C freezer.

  3. Virus titration
    1. Vero E6 cells are split every 2 or 3 days at 1:4 or 1:6 dilutions respectively. Cells are maintained in a CO2 incubator at 5% CO2 and 37 °C. Plan to have confluent T75 flasks ready the afternoon before tittering experiments. Split cells at a 1:4 dilution and then add media containing 200,000 cells to each well of a 12 well plate.
    2. The next day, make dilutions of the virus in DMEM filled wells of a 48 well plate. Dilute 100 µl of sample in 900 µl of DMEM and so on until the sample is appropriately diluted. Plan to infect duplicate wells for each virus, at each dilution. When initially characterizing a SARS-CoV strain try testing the range of 1:10 dilutions from undiluted to 107. Negative control wells are not required.
    3. Prepare overlay medium as described below.
    4. Remove media from the Vero E6 cells and then add 125 µl of each sample dilution to be tested. This must be done rapidly so that the agarose mixture does not solidify before addition to the wells.
    5. Return plates to the CO2 incubator and then gently rock the plate back and forth several times every ten min for 30 min. Observe that rocking is sufficient so that the monolayer remains moist.
    6. Remove the inoculum and add 500 µl of overlay medium per well. When the agarose solidifies add 500 µl of 2% serum culture media per well and return plate to the incubator.
    7. On day three post-infection, remove plates from the incubator and add 1 ml of 25% formaldehyde diluted in PBS to each well. Fix cells for 20 min at room temperature and then suction off liquid using a pipet and pipet-aid. Remove agarose layer using a small spatula, being careful not to accidentally disrupt the monolayer.
    8. Stain cells with 200 µl of 0.1% crystal violet per well for at least 5 min, remove dye, wash once with ddH2O, and count plaques.


      Figure 1. Visualization of plaques Monolayers of indicator cells and dilutions of virus were prepared. Samples were inoculated onto cells and plaques visualized as described above. Wells on right hand side of figure contain no plaques while the upper two rows in the first 2 columns illustrate nearly complete destruction of the monolayer. Clearly delineated plaques are shown in the bottom row.

  4. Quantification of lung viral titer
    1. Viral titers are expressed as Log10 PFU/gram of tissue, calculated using the following equation: Log [(number of plaques x 10dilution factor x 1/volume used to overlay cells x 3)/weight of lungs]. 3 is the volume of PBS used to homogenize the tissue. For example let’s hypothesize that there were 5 plaques in a well that had received a sample that was diluted by a factor of 1000. We add 0.125 L of sample to each well and assume a mean weight of 0.14 g for each mouse lung. The titer is then calculated as follows: Log [(5*103*1/0.125*3)/0.14] = 5.9 Log10PFU/gm

Recipes

  1. Ketamine solution (10 ml)
    1.75 ml ketamine
    0.25 ml xylazine
    8 ml 0.9% sodium chloride irrigation
    Stored at room temperature in a double locked cabinet
  2. Culture media (500 ml)    
    444.5 ml DMEM with Hepes (25 mM)
    20 ml L-Glutamine
    20 ml FBS
    10 ml MEM non-essential amino acids
    0.5 ml gentamicin sulfate
    5 ml penicillin streptomycin
    Stored at 4 °C
  3. Overlay medium
    Note:  Mix equal volumes of a and b immediately before placing on cells.
    1. 2x medium
      2x DMEM (made from powdered DMEM according to the instructions with 500 ml of H2O instead of 1 L)
      20 ml L-Glutamine
      20 ml FBS
      1 ml gentamicin sulfate
      10 ml MEM non-essential amino acids
      5 ml Penicillin Streptomycin
      Incubate at 37 °C on the day of the assay
    2. Agarose
      1.2% agarose, optimized grade in sterile ddH2O. Agarose is autoclaved and stored at room temperature.  On the day of use, agarose is melted using a microwave and placed at 56 °C. It is mixed with 2x media and directly added to wells.

Acknowledgments

This work was supported by grants from the National Institutes of Health (PO1AI060699, RO1AI091322). The protocol described herein was based on the following manuscript: Zhao et al. (2010).

References

  1. Fett, C., DeDiego, M. L., Regla-Nava, J. A., Enjuanes, L. and Perlman, S. (2013). Complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking E protein. J Virol 87(12): 6551-6559.
  2. Netland, J., DeDiego, M. L., Zhao, J., Fett, C., Alvarez, E., Nieto-Torres, J. L., Enjuanes, L. and Perlman, S. (2010). Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease. Virology 399(1): 120-128.
  3. Zhao, J., Zhao, J. and Perlman, S. (2010). T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice. J Virol 84(18): 9318-9325.

简介

当研究病毒感染的动物模型时的两个关键步骤一致地成功感染动物并在整个感染过程中精确测定组织中的病毒滴度。 在这里我们详细讨论如何感染小鼠与SARS-CoV,然后量化病毒在肺中的效价。

关键字:SARS-CoV, 病毒滴定, 病毒感染, 肺的制备

材料和试剂

  1. 小鼠(NCI)(Balb/c或C57BL/6,4周至18+月)
  2. Vero E6细胞(ATCC,目录号:CRL-1586)
  3. 异氟烷(USP吸入蒸气;液体)(NDC:57319-559-06)
  4. Dulbecco's改良的Eagle培养基高葡萄糖(DMEM)(Life Technologies,Gibco ,目录号:11965092)
  5. 100mg/ml ketaset III氯胺酮HCl注射液(USP)(DEA Schedule II药物)(NDC:0856-2013-01)
  6. 100mg/ml注射甲苯噻嗪(Lloyd Laboratories,NADA号:139-236)
  7. 0.9%氯化钠灌注(USP)(Baxter,目录号:2F7124)
  8. 1×Dulbecco磷酸盐缓冲盐水(DPBS)(Life Technologies,Gibco ,目录号:14190-144)
  9. 甲醛溶液(Sigma-Aldrich,目录号:252549)(37重量%,在H 2 O中)
  10. 结晶紫(Sigma-Aldrich,目录号:C0775)
  11. 25mM具有肝的DMEM(Life Technologies,Gibco ,目录号:32430)
  12. 200mM L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  13. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S11150)
  14. MEM非必需氨基酸(Life Technologies,Gibco ,目录号:11140-050)
  15. 50mg/ml硫酸庆大霉素(Lonza,目录号:17-518Z)
  16. 青霉素链霉素(Life Technologies,Gibco ,目录号:15140-122)
  17. DMEM(Life Technologies,Gibco ,目录号:12100-046)
  18. 琼脂糖(优化级)(Research Products International,目录号:A20090)
  19. 氯胺酮溶液(见配方)
  20. 培养基(见配方)
  21. 覆盖媒体(请参阅配方)

设备

  1. 生物安全3级设施的生物安全罩
  2. 吸收垫(Covidien,目录号:949)
  3. 2ml微管(Sarstedt AG,目录号:72.694.006)
  4. 5ml注射器(BD,目录号:309646)
  5. 75cm 2细胞培养瓶(斜面颈部,0.2μM通气帽)(Corning,目录号:430641)
  6. 12孔细胞培养物簇(带盖的平底)(Corning,Costar ,目录号:3513)
  7. 48孔细胞培养物簇(带盖的平底)(Corning,Costar ,目录号:3548)
  8. (Corning,Costar ,目录号:3548)
  9. 35ml封闭组织研磨系统(Fisher Scientific,目录号:02-542-08)
  10. Dessicator(Narang Medical,目录号:P37.1517P)
  11. 纱布海绵(4×4英寸)(NDC的Pro Advantage ,目录号:P157118)
  12. 1ml注射器(BD,目录号:309659)
  13. 精密滑动针(25G×5/8)(BD,目录号:305122)
  14. 喷雾瓶用70%乙醇
  15. 外科剪刀
  16. 聚苯乙烯泡沫
  17. Pipetman P200微量移液器
  18. Pipetman P1000微量移液器
  19. CO <2>孵化器
  20. 移液助剂
  21. 小金属称重刮刀
  22. 镊子
  23. -80°C冰箱

程序

  1. 感染
    1. 在通风橱中安排材料。 当使用SARS-CoV时,所有程序在BSL3实验室的生物安全罩中进行。 当使用异氟烷时,始终在烟雾或生物安全罩中麻醉动物
    2. 在冰上解冻病毒,然后在DMEM中稀释至适当的浓度。每只小鼠接受50μl病毒。
    3. 将几层纱布放在塑料干燥器的底部,随后加入10ml异氟烷。
      注意:确保将胶带贴在塑料干燥器格栅的中心孔上,因为小老鼠有时能够挤过这个孔,并跳入底部的异氟烷填充室。
    4. 加载P200微量移液器与50微升的病毒,然后将其固定在安全的位置,同时将鼠标放在干燥器内的炉排。
    5. 呼吸速率慢后,立即拿起鼠标,把它垂直,然后慢慢递送50微升的病毒到鼠标的两个鼻孔。当小鼠轻度麻醉时,呼吸将足够深,使得鼻孔上的液体应该完全吸入肺中。
      注意:使用移液器吸头的孔口尺寸显着;尖端太小,使得难以顺利地将病毒分配到鼻孔上。

  2. 肺组织的去除和加工
    1. 通过腹膜内注射300μl氯胺酮/赛拉嗪溶液使小鼠安乐死。要将流体注入腹腔内,将鼠标固定在手掌中,并将其水平握住。以与小鼠相切的浅角度插入25G×5/8g针,并慢慢地将氯胺酮/赛拉嗪分配到腹膜腔中。
    2. 一旦小鼠完全麻醉,将其放置在聚苯乙烯泡沫上的吸收剂填充的正方形上,并通过用25G×5/8号针固定每个肢体来固定小鼠。
    3. 使用装满70%乙醇的喷雾瓶对小鼠的毛皮进行消毒
    4. 使用剪刀通过首先从动物的下腹部到喉咙的切口切开腹腔和胸腔。打开腹腔暴露隔膜的下侧。使用剪刀刺穿隔膜。切除鼠标的胸腔,充分暴露心脏和肺。切除心脏远离肺,然后通过切除气管和任何剩余的结缔组织去除肺。
    5. 从胸腔取出肺后,使用装有PBS的5 ml注射器从肺外清洗血液。
    6. 将肺放置在组织研磨机中,并粗略地研磨它们。 加入3毫升PBS的研磨机,继续研磨组织匀浆。
    7. 将1ml匀浆分装到2ml微量管中,置于干冰上,然后转移至-80℃冰箱。

  3. 病毒滴定
    1. Vero E6细胞每2或3天分别以1:4或1:6稀释分开。 将细胞维持在CO 2培养箱中在5%CO 2和37℃下。 计划在滴定实验前下午准备汇合的T75烧瓶。 以1:4稀释分裂细胞,然后向12孔板的每个孔中加入含有200,000个细胞的培养基
    2. 第二天,在48孔板的填充DMEM的孔中进行病毒稀释。稀释100微升样品在900微升的DMEM等,直到样品适当稀释。计划在每个稀释度感染每个病毒的复制孔。当最初表征SARS-CoV毒株时,尝试测试从未稀释到10 7 的1:10稀释度的范围。不需要阴性对照孔。
    3. 如下所述准备覆盖介质。
    4. 从Vero E6细胞中取出培养基,然后加入125μl待测试的每个样品稀释液。这必须快速完成,以便琼脂糖混合物在加入孔之前不会固化
    5. 将平板放回CO 2培养箱中,然后每十分钟轻轻摇动平板数次,每次30分钟。观察到摇摆足以使单层保持湿润
    6. 取出接种物,每孔加入500μl覆盖培养基。当琼脂糖固化时,每孔加入500μl2%血清培养基,并将平板返回培养箱
    7. 在感染后第三天,从培养箱中取出板,并向每个孔中加入1ml稀释于PBS中的25%甲醛。在室温下固定细胞20分钟,然后使用移液管和移液器抽吸液体。使用小刮刀去除琼脂糖层,小心不要意外破坏单层
    8. 染色细胞用200μl的0.1%结晶紫每孔至少5分钟,去除染料,用ddH 2 O洗涤一次,并计数噬斑。


      图1.噬斑的可视化制备指示细胞的单层和病毒的稀释液。 将样品接种到如上所述显现的细胞和噬菌斑上。图右侧的孔不含斑块,而前2列中的上两行说明几乎完全破坏单层。清楚地描绘的斑块显示在底行中。

  4. 肺病毒滴度的定量
    1. 病毒滴度表示为Log 10 PFU /克组织,使用以下公式计算:Log [(噬菌斑数×10)稀释因子 x 1 /用于重叠的体积细胞×3)/肺重量]。 3是用于匀浆组织的PBS的体积。例如,我们假设在一个孔中有5个噬菌斑,其接收到被稀释1000倍的样品。我们向每个孔中加入0.125μL样品,并且对于每只小鼠假定平均重量为0.14g。然后计算滴度,如下:Log [(5×10 3/sup 1 * 1/0.125×3)/0.14] = 5.9 Log 10 PFU/gm

食谱

  1. 氯胺酮溶液(10ml) 1.75 ml氯胺酮 0.25ml甲苯胺
    8毫升0.9%氯化钠灌注
    在室温下储存在双层锁柜
  2. 培养基(500ml)    
    444.5ml具有Hepes(25mM)的DMEM 20ml L-谷氨酰胺 20ml FBS
    10ml MEM非必需氨基酸
    0.5ml庆大霉素硫酸盐 5ml青霉素链霉素 储存在4°C
  3. 覆盖媒介
    注意: 在放入细胞前立即混合等体积的a和b。
    1. 2x介质
      2×DMEM(根据说明书用粉末DMEM由500ml H 2 O而不是1L制备)
      20ml L-谷氨酰胺 20ml FBS
      1 ml硫酸庆大霉素 10ml MEM非必需氨基酸
      5ml青霉素链霉素 在测定当天在37℃下孵育
    2. 琼脂糖
      1.2%琼脂糖,优化级在无菌ddH 2 O中。 将琼脂糖高压灭菌并在室温下贮存。 在使用当天,使用微波将琼脂糖熔化并置于56℃。 将其与2x培养基混合,并直接加入孔中

致谢

这项工作得到国立卫生研究院(PO1AI060699,RO1AI091322)的资助。 本文所述的方案基于以下手稿:Zhao等人(2010)。

参考文献

  1. Fett,C.,DeDiego,M.L.,Regla-Nava,J.A.,Enjuanes,L.and Perlman,S。(2013)。 通过用小鼠免疫在老年小鼠中完全保护免受严重急性呼吸综合征冠状病毒介导的致死性呼吸道疾病 - 适应缺乏E蛋白的病毒。 Virol 87(12):6551-6559。
  2. Netland,J.,DeDiego,M.L.,Zhao,J.,Fett,C.,Alvarez,E.,Nieto-Torres,J.L.,Enjuanes,L.and Perlman, 在E蛋白中缺失的减毒性严重急性呼吸综合征冠状病毒的免疫保护免于致命的呼吸道疾病。 a> Virology 399(1):120-128。
  3. Zhao,J.,Zhao,J.and Perlman,S。(2010)。 T细胞应答对于严重急性呼吸综合征冠状病毒感染的小鼠中的临床疾病和病毒清除是必需的。 84(18):9318-9325。
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引用:Fett, C., Zhao, J. and Perlman, S. (2014). Virus Infection and Titration of SARS-CoV in Mouse Lung. Bio-protocol 4(6): e1084. DOI: 10.21769/BioProtoc.1084.
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