参见作者原研究论文

本实验方案简略版
Jul 2012
Advertisement

本文章节


 

Sendai Virus Propagation Using Chicken Eggs
利用鸡蛋进行仙台病毒的繁殖   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Sendai virus is a member of the family Paramyxoviridae, and an enveloped virus with a negative-stranded RNA genome. Sendai virus is not pathogenic to humans, but for mice and can cause pneumonia in mice. Easy and efficient techniques for propagating Sendai virus are required for studying virus replication, virus-induced innate- and adaptive-immunity, Sendai-virus-based virotherapy and IgA nephropathy. Here, we describe a protocol for Sendai virus propagation using chicken eggs. This traditional protocol enables us to generate a large amount of virus enough for animal experiments as well as cell culture experiments in a relatively inexpensive way.

Keywords: Sendai virus (仙台病毒), Chicken eggs (鸡蛋), Propagation (传播), Harvest (收获), Allantoic fluid (尿囊液), Mouse parainfluenza virus (小鼠副流感病毒)

Background

Sendai virus (SeV) is a mouse parainfluenza virus type 1 that was discovered in Sendai, Japan, in the 1950s (Ishida and Homma, 1978). The virus was once named Hemagglutinating Virus of Japan (HVJ) by the Japanese Society for Virology, but was later termed ‘newborn virus pneumonitis (type Sendai)’ (Kuroya and Ishida, 1953). The name SeV is currently most popular, and now understood to be a pathogen of mice, not humans (Karron RA, 2007). Fukumi et al. first described SeV infections of mice in 1954 (Fukumi et al., 1954). This infection can be subclinical, but SeV is also known as one of the leading causes of pneumonia in certain mouse strains (Fukumi et al., 1954; Parker et al., 1978). SeV is an excellent tool to study the following in the various fields: the pathomechanism of a murine model of IgA nephropathy (Yamashita et al., 2007; Chintalacharuvu et al., 2008), a stimulator of RIG-I/MDA5 in innate immunity (Fensterl et al., 2008; Chattopadhyay et al., 2010, 2011 and 2013; Yamashita et al., 2012a, 2012b and 2013), oncolytic SeV-based virotherapy (Saga and Kaneda, 2015), a respiratory infection (Hermesh et al., 2010 and 2012), and a vector for AIDS vaccine (Ishii and Matano, 2015). SeV is uniquely sensitive to interferon-associated responses, and grows to high titers in both chicken eggs and in FDA-approved mammalian cell lines, an advantage for vaccine production. This protocol provides a method for SeV propagation using chicken eggs. This method can be applied for viral propagation for other viruses such as influenza virus.

Materials and Reagents

  1. 3 ml disposable transfer pipette (Bioland Scientific, catalog number: TPP02-11 )
  2. Centrifuge bottles, 250 ml (Fisher Scientific, catalog number: 05-564-1)
    Manufacturer: Thermo Fisher Scientific, catalog number: 3141-0250 .
  3. Bucket with ice
  4. 25 G 5/8 needle (Fisher Scientific, catalog number: 14-826AA)
    Manufacturer: BD, catalog number: 305122 .
  5. 1 ml syringes (BD, catalog number: 309659 )
  6. 18 G needle (Fisher Scientific, catalog number: 14-826-5G)
    Manufacturer: BD, catalog number: 305195 .
  7. 50 ml syringe (Fisher Scientific, catalog number: 14-955-461 )
  8. Pencil
  9. Push pin (Staples, catalog number: 480117 )
  10. Face mask (Cellucap Manufacturing, catalog number: 1826EL )
  11. Iodine Wipes (Dynarex, catalog number: B003U463PY )
  12. Egg holder/case (provided with embryonated eggs, or you can obtain regular ones from grocery store when you purchase regular/unembryonated eggs)
  13. Chicken embryonated eggs (48 eggs, 9-10 days old) (Charles River, catalog number: 10100332 )
  14. Duco® Cement Multi-Purpose Household Glue (ITW Consumer, Duco Cement, catalog number: 62435 )
  15. Sendai virus stock (ATCC, catalog number: VR-105 or catalog number: VR-907 )
  16. PBS with Ca2+ and Mg2+ (Thermo Fisher Scientific, catalog number: 14040133 )
  17. 70% ethanol
  18. Bleach (Essendant, catalog number: CLO30966CT )
  19. 0.5 M EDTA (Thermo Fisher Scientific, catalog number: R1021 )

Equipment

  1. Pipette (200 μl and 1,000 μl; any products are fine)
  2. Biosafety cabinet (any types are fine if it is BSL2 level)
  3. 1 L Graduated cylinder (Thermo Fisher Scientific, catalog number: 3664-1000 )
  4. Egg Incubator (up to 41 eggs; Farm Innovators, Digital Circulated Air Incubator with Auto Egg Turner, model: 4250 )
  5. Refrigerator/4 °C cold room
  6. High-speed Centrifuge with fixed-angle rotor of 250 ml bottles (Beckman Coulter, model: Avanti® J-E )
  7. Sonic water bath (Skymen Cleaning Equipment, model: JP-008 )
  8. Flashlight (Defiant, catalog number: HD15FL04-3 or Caliburn Lighting, catalog number: PISTOL-TRIPOD-RCH
  9. Scissors (World Precision Instruments, catalog number: 14192-G )
  10. Forceps (World Precision Instruments, catalog number: 501974 )

Procedure

  1. Egg candling on Day 10
    Eliminate infertile and fertile dead eggs by egg candling, mark the position of embryo, and air cell with a pencil (Figure 1). You need a dark room and concentrated light source. The best evidence of a live embryo is well-developed blood vessels.
    Note: The capacity of the egg incubator is 41 eggs. If you have the egg incubator with larger capacity (e.g., 160 eggs), you can order 180 eggs, and handle 160 eggs.


    Figure 1. Chicken egg with air cell. Air cell is visualized by candling (left). Draw a line along the edge of air cell (arrows in left panel) and mark X (right) in the position of the embryo with a pencil under a strong light in dark room.
    Note: This is an example of regular/unembryonated egg. You will see an embryo as a slightly dark spot and red vessels (right) by candling in the embryonated egg on Day 10.

  2. Allantoic inoculation on Day 10
    In a biosafety cabinet, dilute sterile Sendai virus stock to a concentration of 1 x 106 pfu/ml in sterile PBS with Ca2+ and Mg2+. The inoculum is 0.2 ml, containing 2 x 105 pfu. Keep tubes of virus on ice.
    1. Wipe top (air cell side) of egg with 70% ethanol.
    2. Wipe top of egg with iodine and let dry.
    3. Puncture hole with alcohol soaked push pin at the location opposite to the position of the embryo and 10 mm below the air cell edge (Figure 2).
      Note: Blood vessels usually do not reach to this area (10 mm below the air cell edge). Allantoic fluid does not leak from the punctured hole.


      Figure 2. Chicken egg with a puncture. Make a hole with a push pin at the location 10 mm below the air cell edge and opposite to embryo.

    4. Insert 25 G 5/8 needle to the hilt, deliver 0.2 ml inoculum. Do not inject any air.
    5. Seal hole with Duco cement, let dry.
    6. Return eggs to the incubator, turn on the turner.
      Note: The egg incubator contains the function of turning eggs every four hours to cause the yolk to be repositioned away from the shell, making it safe for developing embryos.

  3. Egg candling on Day 12
    Candle the eggs at the end of the day, mark and discard the dead ones. Place all live ones in a refrigerator/cold room. Do not stack them because they need to be cooled quickly for proper contraction of the blood vessels. Keep the eggs in the cold overnight, and process the next day.

  4. Harvest of allantoic fluid on Day 13
    From this step, all procedures need to be performed in a biosafety cabinet. Wearing a face mask is recommended because the virus can cause mild respiratory symptom in humans. Cut off shell that covers the air cell using scissors and forceps. Break the shell membrane, avoid rupturing yolk or blood vessels with an 18 G needle on a 50 ml syringe, and aspirate allantoic fluid (yellowish tinge) (Figure 3). From one large egg 10-15 ml usually can be collected. Watch for yolk (very yellow) and egg white (albumin: clear and very viscous) (Figure 4). The yolk and the egg white with viral inoculation do not show any difference from the ones of regular/unembryonated eggs from grocery store in appearance and smell. If an egg looks nasty (i.e., egg white looks very whitish or yellowish, and/or are stinky), this is a phenomenon of bacterial growth in the egg and the egg should be thrown away after immediate bleaching (disinfecting). Place the collected allantoic fluid in centrifuge tubes (250 ml) on ice immediately.
    Note: On Day 13, yolk is not easily visible. Blood vessels do not reach to the air cell, and will be shrunk after cooling the eggs in the cold room; Red blood cells and shell will spin out with clarification, and not be a problem. However, membrane pieces will co-purify with the virus. Therefore, avoid to remove membrane pieces.


    Figure 3. How to open up an egg. A. Making a hole on the shell over air cell. B. Cutting shell by scissors along but slightly inside of the line of air cell. C. Exposed air cell and intact shell membrane. D. Peeling and removal of shell membrane with forceps completely; E. Collecting allantoic fluid between shell membrane and an embryo (yolk in this picture) with an 18 G needle and 50 ml syringe.
    Note: This is an example of unembryonated egg. You will see an embryo instead of yellow yolk in the actual viral propagation.


    Figure 4. Collected allantoic fluid. Allantoic fluid is transparent or slightly cloudy with yellowish tinge.

  5. Concentration of SeV by centrifugation
    1. Collect allantoic fluid into 250 ml centrifuge bottles (max volume 225 ml) on ice. Balance bottles.
    2. Spin the rotor at 2,600 x g for 25 min to remove the shell. Do not use brake.
    3. Decant fluid into fresh centrifuge bottles that contain 2 ml of 0.5 M EDTA. This will make the pellet easier to be re-suspended later.
    4. Spin the rotor at 27,000 x g for 90 min to obtain the 1st pellet. Decant fluid into a graduated cylinder for the volume measurement, and discard the fluid after immediate bleaching.
    5. Re-suspend the pellet in cold PBS (with Ca2+ and Mg2+) with 1 mM EDTA using a 2-3 ml disposable transfer pipette. Condense multiple bottles into one bottle. Extensive rinsing will be required to re-suspend the first pellet. Bring the volume up to about 200 ml with cold PBS (with Ca2+ and Mg2+) with 1 mM EDTA.
      Note: The purpose of Steps E5 and E6 is to combine multiple pellets to be one single pellet. If you do not have many bottles, you do not need these steps.
    6. Spin the rotor at 27,000 x g for 90 min to obtain the second pellet. Decant the fluid and discard after immediate bleaching.
    7. Re-suspend the pellet in about 2 ml of cold PBS (with Ca2+ and Mg2+) with 1 mM EDTA. Use another 8-10 ml to rinse the remainder, and pool into a tube.
    8. Sonicate (40 kHz frequency and 35 W ultrasonic power) final suspension for 2 min at room temperature in a sonic water bath to break apart viral chunk.
    9. Aliquot and freeze them at -80 °C. Make and freeze also two small aliquots for testing viral titer.

Notes

This protocol needs an appropriate facility (Biosafety level 2) and an approved Institutional Biosafety Committee (IBC) protocol.

Acknowledgments

Dr. Yamashita was supported by American Heart Association Scientist Development Grant (17SDG33660947) and University of California Los Angeles (UCLA) Clinical and Translational Science Institute (CTSI) Grant (UL1TR001881), and is supported by UCLA CTSI KL2 grant (KL2TR001882) and Cedars-Sinai CTSI Clinical Scholars Grant. We would like to thank Dr. Steven Emancipator for the mentoring in the early stage of Dr. Yamashita's career development and Dr. John Nedrud for previous collaboration. The protocol was adapted from our previous work (Yamashita et al., 2007; Chintalacharuvu et al., 2008).

Competing interests

Authors declare no conflicts of interest or competing interests.

References

  1. Chattopadhyay, S., Fensterl, V., Zhang, Y., Veleeparambil, M., Yamashita, M. and Sen, G. C. (2013). Role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by Sendai virus. J Virol 87(1): 16-24.
  2. Chattopadhyay, S., Marques, J. T., Yamashita, M., Peters, K. L., Smith, K., Desai, A., Williams, B. R. and Sen, G. C. (2010). Viral apoptosis is induced by IRF-3-mediated activation of Bax. EMBO J 29(10): 1762-1773.
  3. Chattopadhyay, S., Yamashita, M., Zhang, Y. and Sen, G. C. (2011). The IRF-3/Bax-mediated apoptotic pathway, activated by viral cytoplasmic RNA and DNA, inhibits virus replication. J Virol 85(8): 3708-3716.
  4. Chintalacharuvu, S. R., Yamashita, M., Bagheri, N., Blanchard, T. G., Nedrud, J. G., Lamm, M. E., Tomino, Y. and Emancipator, S. N. (2008). T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy. Clin Exp Immunol 153(3): 456-462.
  5. Fensterl, V., White, C. L., Yamashita, M. and Sen, G. C. (2008). Novel characteristics of the function and induction of murine p56 family proteins. J Virol 82(22): 11045-11053.
  6. Fukumi, H., Nishikawa, F. and Kitayama, T. (1954). A pneumotropic virus from mice causing hemagglutination. Jpn J Med Sci Biol 7(4): 345-363.
  7. Hermesh, T., Moltedo, B., Moran, T. M. and Lopez, C. B. (2010). Antiviral instruction of bone marrow leukocytes during respiratory viral infections. Cell Host Microbe 7(5): 343-353.
  8. Hermesh, T., Moran, T. M., Jain, D. and Lopez, C. B. (2012). Granulocyte colony-stimulating factor protects mice during respiratory virus infections. PLoS One 7(5): e37334.
  9. Ishida, N. and Homma, M. (1978). Sendai virus. Adv Virus Res 23: 349-383.
  10. Ishii, H. and Matano, T. (2015). Development of an AIDS vaccine using Sendai virus vectors. Vaccine 33(45): 6061-6065.
  11. Karron RA, C. P. (2007). Parainfluenza Viruses (5th Edition). Philadelphia, PA: Lippincott Williams and Wilkins.
  12. Kuroya, M. and Ishida, N. (1953). Newborn virus pneumonitis (type Sendai). II. The isolation of a new virus possessing hemagglutinin activity. Yokohama Med Bull 4(4): 217-233.
  13. Parker, J. C., Whiteman, M. D. and Richter, C. B. (1978). Susceptibility of inbred and outbred mouse strains to Sendai virus and prevalence of infection in laboratory rodents. Infect Immun 19(1): 123-130.
  14. Saga, K. and Kaneda, Y. (2015). Oncolytic Sendai virus-based virotherapy for cancer: recent advances. Oncolytic Virother 4: 141-147.
  15. Yamashita, M., Chattopadhyay, S., Fensterl, V., Saikia, P., Wetzel, J. L. and Sen, G. C. (2012a). Epidermal growth factor receptor is essential for Toll-like receptor 3 signaling. Sci Signal 5(233): ra50.
  16. Yamashita, M., Chattopadhyay, S., Fensterl, V., Zhang, Y. and Sen, G. C. (2012b). A TRIF-independent branch of TLR3 signaling. J Immunol 188(6): 2825-2833.
  17. Yamashita, M., Chintalacharuvu, S. R., Kobayashi, N., Nedrud, J. G., Lamm, M. E., Tomino, Y. and Emancipator, S. N. (2007). Analysis of innate immune responses in a model of IgA nephropathy induced by Sendai virus. Contrib Nephrol 157: 159-163.
  18. Yamashita, M., Millward, C. A., Inoshita, H., Saikia, P., Chattopadhyay, S., Sen, G. C. and Emancipator, S. N. (2013). Antiviral innate immunity disturbs podocyte cell function. J Innate Immun 5(3): 231-241.

简介

仙台病毒是家族副粘病毒科的成员,以及具有负链RNA基因组的包膜病毒。 仙台病毒对人类无致病性,但对小鼠而言可导致小鼠肺炎。 传播仙台病毒的简便有效的技术是研究病毒复制,病毒诱导的先天免疫和适应性免疫,基于仙台病毒的病毒疗法和IgA肾病所必需的。 在这里,我们描述了使用鸡蛋传播仙台病毒的协议。 这种传统的方案使我们能够以相对便宜的方式产生足够的大量病毒用于动物实验以及细胞培养实验。

【背景】仙台病毒(SeV)是一种小鼠副流感病毒1型,于20世纪50年代在日本仙台发现(Ishida和Homma,1978)。该病毒曾被日本病毒学会命名为日本血凝病毒(HVJ),后来被称为“新生病毒性肺炎(仙台型)”(Kuroya和Ishida,1953)。 SeV这个名字目前最受欢迎,现在被认为是老鼠的病原体,而不是人类(Karron RA,2007)。 Fukumi 等人首次描述了1954年小鼠的SeV感染(Fukumi et al。,1954)。这种感染可能是亚临床的,但SeV也被认为是某些小鼠品系中肺炎的主要原因之一(Fukumi et al。,1954; Parker et al。, 1978年)。 SeV是研究以下各个领域的优秀工具:IgA肾病小鼠模型的病理机制(Yamashita et al。,2007; Chintalacharuvu et al。, 2008),RIG-I / MDA5在先天免疫中的刺激物(Fensterl et al。,2008; Chattopadhyay et al。,2010,2011 and 2013; Yamashita et al。,2012a,2012b和2013),基于溶瘤性SeV的病毒疗法(Saga和Kaneda,2015),呼吸道感染(Hermesh et al。,2010和2012),艾滋病疫苗的载体(Ishii和Matano,2015)。 SeV对干扰素相关反应特别敏感,并且在鸡蛋和FDA批准的哺乳动物细胞系中生长至高滴度,这是疫苗生产的优势。该协议提供了使用鸡蛋进行SeV传播的方法。该方法可用于其他病毒如流感病毒的病毒繁殖。

关键字:仙台病毒, 鸡蛋, 传播, 收获, 尿囊液, 小鼠副流感病毒

材料和试剂

  1. 3毫升一次性移液管(Bioland Scientific,目录号:TPP02-11)
  2. 离心瓶250毫升(Fisher Scientific,目录号:05-564-1)
    制造商:Thermo Fisher Scientific,目录号:3141-0250。
  3. 用冰桶
  4. 25 G 5/8针(Fisher Scientific,目录号:14-826AA)
    制造商:BD,目录号:305122。
  5. 1毫升注射器(BD,目录号:309659)
  6. 18 G针(Fisher Scientific,目录号:14-826-5G)
    制造商:BD,目录号:305195。
  7. 50 ml注射器(Fisher Scientific,目录号:14-955-461)
  8. 铅笔
  9. 推针(Staples,目录号:480117)
  10. 面罩(Cellucap Manufacturing,目录号:1826EL)
  11. 碘擦(Dynarex,目录号:B003U463PY)
  12. 蛋架/盒子(提供含胚蛋,或者当您购买常规/未经加工的鸡蛋时,您可以从杂货店获得常规鸡蛋)
  13. 鸡胚鸡蛋(48个鸡蛋,9-10天)(Charles River,目录号:10100332)
  14. Duco ®水泥多用途家用胶水(ITW Consumer,Duco Cement,目录号:62435)
  15. 仙台病毒库存(ATCC,目录号:VR-105或目录号:VR-907)
  16. 具有Ca 2 + 和Mg 2+ 的PBS(Thermo Fisher Scientific,目录号:14040133)
  17. 70%乙醇
  18. Bleach(Essendant,产品目录号:CLO30966CT)
  19. 0.5 M EDTA(Thermo Fisher Scientific,目录号:R1021)

设备

  1. 移液器(200μl和1,000μl;任何产品都很好)
  2. 生物安全柜(如果是BSL2级别,任何类型都可以)
  3. 1 L量筒(赛默飞世尔科技,目录号:3664-1000)
  4. 蛋孵化器(最多41个鸡蛋;农场创新者,带自动蛋转子的数字循环空气孵化器,型号:4250)
  5. 冰箱/ 4°C冷室
  6. 高速离心机,带250毫升瓶的固定角转子(Beckman Coulter,型号:Avanti ® J-E)
  7. 声波水浴(Skymen清洁设备,型号:JP-008)
  8. 手电筒(Defiant,目录号:HD15FL04-3或Caliburn Lighting,目录号:PISTOL-TRIPOD-RCH) 
  9. 剪刀(世界精密仪器,目录号:14192-G)
  10. 镊子(World Precision Instruments,目录号:501974)

程序

  1. 第10天蛋蛋糕
    通过蛋清消除不育和可育的死蛋,用铅笔标记胚胎的位置和空气细胞(图1)。你需要一个黑暗的房间和集中的光源。活胚胎的最佳证据是发育良好的血管。
    注意:鸡蛋培养箱的容量为41个鸡蛋。如果您有更大容量的鸡蛋孵化器(例如,160个鸡蛋),您可以订购180个鸡蛋,并处理160个鸡蛋。


    图1.带有气囊的鸡蛋。通过对光(左)可视化气囊。沿着气室边缘画一条线(左图中的箭头),并在黑暗的房间里用强光照下铅笔在胚胎的位置标记X(右)。
    注意:这是常规/非胚胎蛋的一个例子。在第10天,你会看到一个胚胎是一个略带黑色的斑点和红色的血管(右),在胚胎的蛋中进行光照。

  2. 第10天尿囊接种
    在生物安全柜中,将无菌的仙台病毒原种稀释至无菌PBS中的浓度为1×10 6 pfu / ml,其中Ca 2+ 和Mg 2+ < / SUP>。接种物为0.2ml,含有2×10 5 p pfu。将病毒管放在冰上。
    1. 用70%乙醇擦拭蛋的顶部(气室侧)。
    2. 用碘擦拭鸡蛋顶部并让其干燥。
    3. 用酒精浸泡推针在与胚胎位置相对的位置和气囊边缘下方10 mm的位置(图2)。
      注意:血管通常不会到达该区域(气囊边缘下方10 mm)。尿囊液不会从穿孔中漏出。


      图2.带有穿孔的鸡蛋。在气囊边缘下方10 mm处与胚胎相对的位置用推针打孔。

    4. 将25 G 5/8针头插入刀柄,输送0.2 ml接种物。不要注入任何空气。
    5. 用Duco水泥密封孔,让其干燥。
    6. 将鸡蛋送回培养箱,打开翻转器。
      注意:鸡蛋孵化器包含每四个小时翻蛋的功能,使蛋黄重新定位于壳外,使胚胎发育安全。

  3. 第12天蛋蛋糕
    在一天结束时给鸡蛋上蜡,标记并丢弃死鸡蛋。将所有现场的放在冰箱/冷藏室。不要堆叠它们,因为它们需要快速冷却以适当收缩血管。将鸡蛋放在寒冷的夜间,然后在第二天处理。

  4. 第13天收获尿囊液
    从这一步骤开始,所有程序都需要在生物安全柜中进行。建议戴口罩,因为病毒会在人体内引起轻微的呼吸道症状。使用剪刀和镊子切断覆盖气囊的外壳。打破壳膜,避免在50 ml注射器上用18 G针头破坏卵黄或血管,并吸入尿囊液(淡黄色)(图3)。从一个大的鸡蛋通常可以收集10-15毫升。注意蛋黄(非常黄色)和蛋白(白蛋白:透明且非常粘稠)(图4)。具有病毒接种的蛋黄和蛋白在外观和气味方面与来自杂货店的常规/未发芽蛋没有任何差别。如果一个鸡蛋看起来很讨厌(即,蛋白看起来很白或淡黄色,和/或很臭),这是鸡蛋中细菌生长的现象,鸡蛋应该在立即漂白后扔掉(消毒)。将收集的尿囊液立即置于冰上的离心管(250 ml)中。
    注意:在第13天,蛋黄不容易看见。血管不会到达气囊,冷却后的鸡蛋冷却后会缩小;红细胞和外壳会随着澄清而旋转出来,而不是问题。然而,膜片将与病毒共同纯化。因此,避免去除膜片。


    图3.如何打开鸡蛋。 A.在气囊上打开一个洞。 B.用剪刀剪切外壳,但在气囊线的内侧稍微切割。 C.暴露的空气细胞和完整的壳膜。 D.用镊子完全剥离和去除壳膜; E.用18G针头和50ml注射器收集壳膜和胚胎(图中的蛋黄)之间的尿囊液。
    注意:这是一个非胚胎蛋的例子。在实际病毒传播过程中,你会看到胚胎而不是黄色蛋黄。


    图4.收集的尿囊液。尿囊液透明或略带浑浊,带黄色调。

  5. 通过离心浓缩SeV
    1. 在冰上将尿囊液收集到250ml离心瓶(最大体积225ml)中。平衡瓶。
    2. 将转子以2,600 x g 旋转25分钟以除去壳。不要使用刹车。
    3. 将液体倒入装有2毫升0.5 M EDTA的新鲜离心瓶中。这将使颗粒更容易在以后重新悬浮。
    4. 以27,000 x g 旋转转子90分钟以获得1 st 颗粒。将液体倒入量筒中进行体积测量,并在立即漂白后丢弃液体。
    5. 使用2-3ml一次性移液管将沉淀重悬于含有1mM EDTA的冷PBS(具有Ca 2 + 和Mg 2 + )中。将多个瓶子冷凝成一个瓶子。需要进行大量冲洗以重新悬浮第一颗粒。用冷PBS(含Ca 2+ 和Mg 2 + )和1 mM EDTA将体积增加至约200 ml。
      注意:步骤E5和E6的目的是将多个颗粒组合成一个颗粒。如果您没有很多瓶子,则不需要这些步骤。
    6. 以27,000 x g 旋转转子90分钟以获得第二颗粒。立即漂白后倒出液体并丢弃。
    7. 用1mM EDTA将沉淀重新悬浮于约2ml冷PBS(具有Ca 2 + 和Mg 2 + )中。再用8-10毫升冲洗剩余物,然后装入管中。
    8. 超声处理(40kHz频率和35W超声功率)在室温下在声波水浴中最终悬浮2分钟以破坏病毒块。
    9. 将其等分并在-80℃下冷冻。制作并冷冻两个小等分试样以测试病毒滴度。

笔记

该协议需要适当的设施(生物安全级别2)和经批准的机构生物安全委员会(IBC)协议。

致谢

Yamashita博士得到美国心脏协会科学家发展基金会(17SDG33660947)和加州大学洛杉矶分校(UCLA)临床和转化科学研究所(CTSI)资助(UL1TR001881)的支持,并得到加州大学洛杉矶分校CTSI KL2资助(KL2TR001882)和雪松的支持。 -Sinai CTSI临床学者补助金。我们要感谢Steven Emancipator博士在Yamashita博士职业发展早期的指导和John Nedrud博士之前的合作。该协议改编自我们以前的工作(Yamashita et al。,2007; Chintalacharuvu et al。,2008)。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Chattopadhyay,S.,Fensterl,V.,Zhang,Y.,Veleeparambil,M.,Yamashita,M。和Sen,G。C.(2013)。 干扰素调节因子3介导的细胞凋亡在仙台病毒持续感染的建立和维持中的作用。 J Virol 87(1):16-24。
  2. Chattopadhyay,S.,Marques,J.T.,Yamashita,M.,Peters,K.L.,Smith,K.,Desai,A.,Williams,B.R。和Sen,G.C。(2010)。 通过IRF-3介导的Bax激活诱导病毒性凋亡。 EMBO J 29(10):1762-1773。
  3. Chattopadhyay,S.,Yamashita,M.,Zhang,Y。和Sen,G。C.(2011)。 IRF-3 / Bax介导的细胞凋亡途径,由病毒细胞质RNA和DNA激活,抑制病毒复制。 J Virol 85(8):3708-3716。
  4. Chintalacharuvu,S.R.,Yamashita,M.,Bagheri,N.,Blanchard,T.G。,Nedrud,J.G.,Lamm,M.E.,Tomino,Y。和Emancipator,S.N。(2008)。 T细胞因子极性作为免疫球蛋白A(IgA)糖基化的决定因素和实验性IgA肾病的严重程度。 Clin Exp Immunol 153(3):456-462。
  5. Fensterl,V.,White,C.L.,Yamashita,M。和Sen,G.C。(2008)。 小鼠p56家族蛋白功能和诱导的新特征。 J Virol 82(22):11045-11053。
  6. Fukumi,H.,Nishikawa,F。和Kitayama,T。(1954)。 引起血细胞凝集的小鼠致肺炎病毒。 Jpn J Med Sci Biol 7(4):345-363。
  7. Hermesh,T.,Moltedo,B.,Moran,T。M.和Lopez,C.B。(2010)。 呼吸道病毒感染期间骨髓白细胞的抗病毒指导。 细胞宿主微生物 7(5):343-353。
  8. Hermesh,T.,Moran,T。M.,Jain,D。和Lopez,C.B。(2012)。 粒细胞集落刺激因子在呼吸道病毒感染期间保护小鼠。 PLoS One 7(5):e37334。
  9. Ishida,N。和Homma,M。(1978)。 仙台病毒。 Adv Virus Res 23:349- 383。
  10. Ishii,H。和Matano,T。(2015)。 使用仙台病毒载体开发艾滋病疫苗。 疫苗 33(45):6061-6065。
  11. Karron RA,C。P.(2007)。 Parainfluenza病毒(第5版)。宾夕法尼亚州费城:Lippincott Williams和Wilkins。
  12. Kuroya,M.和Ishida,N。(1953)。 新生儿病毒性肺炎(仙台类型)。 II。分离出一种具有血凝素活性的新病毒。 Yokohama Med Bull 4(4):217-233。
  13. Parker,J.C.,Whiteman,M。D. and Richter,C.B。(1978)。 近交和远交小鼠品种对仙台病毒的易感性和实验室啮齿动物的感染率。 感染免疫 19(1):123-130。
  14. Saga,K。和Kaneda,Y。(2015)。 基于溶瘤性仙台病毒的癌症病毒疗法:近期进展。 Oncolytic Virother 4:141-147。
  15. Yamashita,M.,Chattopadhyay,S.,Fensterl,V.,Saikia,P.,Wetzel,J.L。和Sen,G.C。(2012a)。 表皮生长因子受体对Toll样受体3信号传导至关重要。 科学信号 5(233):ra50。
  16. Yamashita,M.,Chattopadhyay,S.,Fensterl,V.,Zhang,Y。和Sen,G.C。(2012b)。 与TRIF无关的TLR3信号分支。 J Immunol 188(6):2825-2833。
  17. Yamashita,M.,Chintalacharuvu,S.R.,Kobayashi,N.,Nedrud,J.G.,Lamm,M.E.,Tomino,Y。和Emancipator,S.N。(2007)。 仙台病毒诱导的IgA肾病模型中的先天免疫反应分析。 Contrib Nephrol 157:159-163。
  18. Yamashita,M.,Millward,C.A.,Inoshita,H.,Saikia,P.,Chattopadhyay,S.,Sen,G.C。和Emancipator,S.N。(2013)。 抗病毒先天免疫干扰足细胞功能。 J Innate Immun 5(3):231-241。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tatsumoto, N., Arditi, M. and Yamashita, M. (2018). Sendai Virus Propagation Using Chicken Eggs. Bio-protocol 8(18): e3009. DOI: 10.21769/BioProtoc.3009.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。