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Human Astrovirus Propagation, Purification and Quantification
人星状病毒繁殖、纯化和量化   

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Abstract

Astrovirus are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. Here, we describe astrovirus propagation, purification and titration. The Caco-2 human intestinal adenocarcinoma cell line is most widely used for studying astrovirus, although other cell lines, such as 293, T84 and LLC-MK2 can be used for propagation. However, Caco2 cells are desirable for their ability to form a differentiated intestinal epithelium, mimicking the human intestine and providing a realistic model for astrovirus growth and propagation.

Keywords: Astrovirus(星状病毒), Propagation(传播), Purification(净化), Titration(滴定法), Intestine(肠)

Materials and Reagents

  1. Caco2 cell line (ATCC, catalog number: HTB-37 )
  2. MEM (Mediatech, Cellgro®, catalog number: 10-010-CV , or equivalent)
  3. Glutamax (Life Technologies, Gibco®, catalog number: 35050-061 , or equivalent),
  4. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360-070 , or equivalent)
  5. 10% heat-inactivated FBS
  6. Porcine type 1x trypsin (Sigma-Aldrich, catalog number: T-0303 , or equivalent)
  7. MgCl2
  8. Sucrose
  9. Phosphate buffered saline (PBS)
  10. Bovine serum albumin Fraction V (BSA) (Life Technologies, Gibco®, catalog number: 15260-037 )
  11. Formaldehyde (Polysciences, catalog number: 18814 )
  12. TritonX-100 (Sigma-Aldrich, catalog number: 9002-93-1 )
  13. Normal goat serum (Sigma-Aldrich, catalog number: 191356 )
  14. 4',6-diamidino-2-phenylindole (DAPI) (Life Technologies, Molecular Probes®, catalog number: D1306 )
  15. Astrovirus 8E7 mouse monoclonal antibody (hybridoma cell line) (ATCC, catalog number: HB-11945 , or equivalent)
  16. Fluorescent-conjugated anti-mouse IgG antibody [Life Technologies, Alexa Fluor® 488 Goat Anti-Mouse (H+L) Antibody, catalog number: A11001 , or equivalent]
  17. Astrovirus (not commercially available)
  18. Bleach (clorox or stored brand equivalent)
  19. Virkon S (DuPont)
  20. Caco2 cell culture medium (see Recipes)
  21. Serum-free (SF) Caco2 medium (see Recipes)
  22. TN buffer (see Recipes)

Equipment

  1. Biosafety cabinet
  2. Gloves
  3. Labcoat
  4. Beckman ultracentrifuge tubes (ultra-clear 9/16 x 3 ½ in) (Beckman Coulter, catalog number: 344059 )
  5. Beckman ultracentrifuge (that can reach 34,000 rpm and 4 ºC)
  6. SW41 rotor
  7. Pierce Extra-Strength Slide-A-Lyzer 10K molecular weight cassette (Pierce, catalog number: 66383 or 66380 )
  8. Laminar flow hood
  9. Syringe
  10. Liquid nitrogen
  11. Water bath

Procedure

  1. Propagating human Astrovirus
    1. Seed T-75 flask(s) with 2.5-5 x 106 Caco2 cells in cell culture medium. Grow at 37 ºC in 5% CO2 for 3-5 days until cells reach 100% confluence.
    2. Rinse confluent Caco2 cells 1x with 3-4 ml PBS.
    3. Incubate cells for 1 h at 37 ºC, 5% CO2 in SF Caco2 Medium containing 5 µg/ml porcine trypsin.
    4. All subsequent steps should be performed in a certified biosafety cabinet by personnel wearing disposable gloves and a labcoat working under BL2 conditions.
    5. Remove media and infect cells with astrovirus in 5 ml SF Caco2 Medium with 5 µg/ml porcine trypsin for 90 min at 37 ºC, 5% CO2 (see Notes 1 and 2).
    6. Remove the infection media and replace with 6 to 7 ml SF Caco2 Medium with 10 µg/ml porcine trypsin (see Note 3). Use extreme caution as the cells will begin to detach due to the trypsin.
    7. Incubate at 37 ºC, 5% CO2 for 3 to 4 days, then collect supernatant and cells.
    8. To increase yield, sonicate 4 x 15 sec. Alternatively, pellet cells by centrifuging for 5 min at 4,000 rpm. Remove all but 1 ml of supernatant and freeze-thaw the cell pellet 3 times by freezing in liquid nitrogen and thawing at 37 ºC. Centrifuge again to pellet cell debris and combine supernatants.
    9. Aliquot the virus and stored at -80 ºC.

  2. Purification of Astrovirus
    1. Pre-clear astrovirus solution by centrifuging at 4,000 rpm to pellet cellular debris.
    2. For each tube
      1. Pipet 4 ml of 15% sucrose (w/v) in TN buffer into a 12 ml Beckman ultra-clear 9/16 x 3 ½ in ultracentrifuge tube.
      2. Underlay with 2 ml of 50% sucrose (w/v) in TN buffer.
      3. Pipet approximately 6 ml of pre-cleared astrovirus supernatant (or PBS/TN buffer if a balance is needed) on top of the 15% sucrose layer (Figure 1A).
    3. Spin in SW41 ultracentrifuge rotor at 34,000 rpm for 3 h at 4 °C.
    4. Remove virus by puncturing the side of the tube with a syringe just under the virus band (white cloudy band at the 15%/50% interface; Figure 1B; see Note 4).
    5. Insert virus into Slide-A-Lyzer 10K dialysis cassette. Remove excess air in the cassette.
    6. Dialyze virus in PBS + 10 mM MgCl2 for at least 4 h with 2 buffer changes (2 L total) or overnight in 2 L buffer at 4 ºC.
    7. Remove virus using a syringe. Aliquot and freeze at -80 ºC.


      Figure 1. Schematic of sucrose gradient. Schematic of gradient loading (A) and the viral band after centrifugation (B).

  3. Astrovirus Fluorescent Focus Assay
    1. Seed a 96-well plate with 2 x 104 Caco2 cells/well. Grow 3-4 days until cells reach 100% confluence.
    2. Gently rinse cells 2x with sterile PBS. Trypsinize and count 2-3 wells to calculate the number of cells/well.
    3. Add 100 µl of SF Caco2 media (supplemented with 0.3% BSA) and incubate for 1 h at 37 °C, 5% CO2.
    4. Prepare ten-fold viral dilutions in SF Caco2 media containing 0.3% BSA.
    5. Remove media from cells and add 100 µl of serial dilutions to cells. Each sample should be assayed in triplicate.
    6. Incubate 1 h at 37 °C, 5% CO2. Remove media and replace with SF Caco2 media containing 0.3% BSA.
    7. Incubate plate at least 10 h at 37 °C, 5% CO2, but not more than 24 h.
    8. Remove media. Rinse cells gently 3x with PBS.
    9. Fix in 100 µl of 4% formaldehyde/PBS at room temperature for 20 min.
    10. Rinse 3x with ~100-200 µl of PBS. All rinses should be performed with this approximate volume.
    11. Permeabilize for 10 min at room temperature with 100 µl of PBS containing 0.5% TritonX-100.
    12. Rinse 3x with PBS.
    13. Block 1 h in 100 µl of 5% normal goat serum in PBS at room temperature. Plate can be gently rocked if desired.
    14. Rinse 3x with PBS.
    15. Incubate with primary mouse anti-HAstV-1 capsid protein (8E7) 1:100 in 50 µl 1% normal goat serum in PBS for 1-2 h at room temperature or overnight at 4 °C. Plate can be gently rocked if desired.
    16. Rinse 3x with PBS.
    17. Incubate with secondary goat anti-mouse AlexaFluor 488 1:100 in 50 µl 1% normal goat serum in PBS+ 1 µg/ml DAPI. Keep plate away from light from this step forward. Plate can be gently rocked if desired.
    18. Rinse 3x with PBS. Add ~200 µl PBS to each well and visualize on fluorescent microscope (Figure 2).
    19. Calculate fluorescent focus units per ml (FFU/ml): %FITC+ cells x average number of cells/well x dilution factor = FFU/Ml


      Figure 2. Example of Astrovirus staining. IFA staining of uninfected (left) and astrovirus infected (right) Caco2 cells.Astrovirus capsid is shown in green, and cell nuclei in blue.

Notes

  1. For propagating astrovirus stocks that were made following this protocol, infect with an MOI: 1 (if possible). If using fecal filtrate or stocks that have not been grown in the presence of porcine trypsin, pre-incubate filtrate/stock with 10 µg/ml porcine trypsin for 1 h before adsorption onto Caco2 cells.
  2. During the 90 min infection, rock cells every 15 min.
  3. Inactivate virus in inoculum by incubating in 10% vol/vol bleach for at least 30 min before disposal; autoclaving inoculum; or incubating in 1% vol/vol Virkon S for 30 min.
  4. Visualization of the viral band can be increased by either placing a black background behind the ultracentrifuge tube, or by turning the lights off in the hood/room and shining light at the band.

Recipes

  1. Caco2 cell culture medium
    MEM supplemented with:
    1% glutamax
    1% sodium pyruvate
    10% heat-inactivated FBS
  2. Serum-free (SF) Caco2 medium
    MEM supplemented with:
    1% glutamax
    1% sodium pyruvate
  3. TN buffer
    50 mM Tris (pH 7.5)
    100 mM NaCl
    Sterilized

Acknowledgments

This protocol was adapted from the previous publications DuBois et al. (2013); Moser and Schultz-Cherry (2008); and Willcocks et al. (1990). Funding for this research was provided by the Children’s Infection Defense Center, the Hartwell Foundation, and the American Lebanese Syrian Associated Charities and St Jude Children’s Research Hospital.

References

  1. DuBois, R. M., Freiden, P., Marvin, S., Reddivari, M., Heath, R. J., White, S. W. and Schultz-Cherry, S. (2013). Crystal structure of the avian astrovirus capsid spike. J Virol 87(14): 7853-7863.
  2. Moser, L. A. and Schultz-Cherry, S. (2008). Suppression of astrovirus replication by an ERK1/2 inhibitor. J Virol 82(15): 7475-7482.
  3. Willcocks, M., Carter, M., Laidler, F. and Madeley, C. (1990). Growth and characterisation of human faecal astrovirus in a continuous cell line. Arch Virol 113(1-2): 73-81.

简介

星状病毒是小的,未发育的单链RNA病毒,其在多种哺乳动物和鸟类中引起腹泻。 在这里,我们描述星状病毒繁殖,纯化和滴定。 Caco-2人肠腺癌细胞系最广泛地用于研究星形病毒,尽管其它细胞系,例如293,T84和LLC-MK2可用于繁殖。 然而,Caco2细胞是其形成分化的肠上皮的能力,模拟人的肠并且为星状病毒生长和繁殖提供现实模型所需的。

关键字:星状病毒, 传播, 净化, 滴定法, 肠

材料和试剂

  1. Caco2细胞系(ATCC,目录号:HTB-37)
  2. MEM(Mediatech,Cellgro ,目录号:10-010-CV或等同物)
  3. Glutamax(Life Technologies,Gibco ,目录号:35050-061或等价物),
  4. 丙酮酸钠(Life Technologies,Gibco ,目录号:11360-070或等同物)
  5. 10%热灭活的FBS
  6. 猪类型1x胰蛋白酶(Sigma-Aldrich,目录号:T-0303或等价物)
  7. MgCl 2
  8. 蔗糖
  9. 磷酸盐缓冲盐水(PBS)
  10. 牛血清白蛋白级分V(BSA)(Life Technologies,Gibco ,目录号:15260-037)
  11. 甲醛(Polysciences,目录号:18814)
  12. TritonX-100(Sigma-Aldrich,目录号:9002-93-1)
  13. 正常山羊血清(Sigma-Aldrich,目录号:191356)
  14. 4',6-二脒基-2-苯基吲哚(DAPI)(Life Technologies,Molecular Probes ,目录号:D1306)
  15. 星状病毒8E7小鼠单克隆抗体(杂交瘤细胞系)(ATCC,目录号:HB-11945或等同物)
  16. 荧光缀合的抗小鼠IgG抗体[Life Technologies,Alexa Fluor <488山羊抗小鼠(H + L)抗体,目录号:A11001或等同物]
  17. 星状病毒(不可商购)
  18. 漂白剂(clorox或存储品牌等同物)
  19. Virkon S(杜邦)
  20. Caco2细胞培养基(参见配方)
  21. 无血清(SF)Caco2培养基(参见配方)
  22. TN缓冲区(参见配方)

设备

  1. 生物安全柜
  2. 手套
  3. Labcoat
  4. Beckman超速离心管(超清洁9/16×3 1/2英寸)(Beckman Coulter,目录号:344059)
  5. Beckman超速离心机(可达到34,000 rpm和4℃)
  6. SW41转子
  7. Pierce Extra-Strength Slide-A-Lyzer 10K分子量盒(Pierce,目录号:66383或66380)
  8. 层流罩
  9. 注射器
  10. 液氮
  11. 水浴

程序

  1. 增殖人星状病毒
    1. 种子在细胞培养基中具有2.5-5×10 6个Caco2细胞的T-75烧瓶。在37℃在5%CO 2中生长3-5天,直到细胞达到100%汇合。
    2. 用3-4ml PBS冲洗汇合的Caco2细胞1×
    3. 在37℃,5%CO 2下在含有5μg/ml猪胰蛋白酶的SF Caco2培养基中孵育细胞1小时。
    4. 所有后续步骤都应在经认证的生物安全柜中由佩戴一次性手套和实验室工作的人员在BL2条件下进行。
    5. 在37℃,5%CO 2下,用5μg/ml猪胰蛋白酶在5ml SF Caco2培养基中除去培养基并感染细胞用星形病毒感染90分钟(参见注释1和2)。
    6. 取出感染培养基,更换为含有10μg/ml猪胰蛋白酶的6〜7 ml SF Caco2培养基(见注3)。请特别小心,因为胰蛋白酶会使细胞开始分离。
    7. 在37℃,5%CO 2孵育3至4天,然后收集上清液和细胞。
    8. 为了增加产量,超声处理4×15秒。 或者,通过在4,000rpm离心5分钟沉淀细胞。 除去所有1毫升的上清液和冷冻解冻细胞沉淀3次,在液氮中冷冻和解冻在37℃。 再次离心以沉淀细胞碎片并合并上清液
    9. 等分病毒并储存在-80℃。

  2. 星状病毒的纯化
    1. 通过在4,000rpm离心以沉淀细胞碎片来预清除星状病毒溶液。
    2. 对于每个管
      1. 吸取4毫升15%蔗糖(w/v)在TN缓冲液中的12毫升贝克曼超清9/16×3/2超速离心管。
      2. 用2ml 50%蔗糖(w/v)在TN缓冲液中的底层
      3. 在15%蔗糖层顶部吸取约6ml预清除的星状病毒上清液(或如果需要平衡,则为PBS/TN缓冲液)(图1A)。
    3. 在SW41超速离心机转子中以34,000rpm在4℃下离心3小时
    4. 通过用刚好在病毒带下的注射器穿刺管的侧面来除去病毒(在15%/50%界面处的白色混浊带;图1B;参见注释4)。
    5. 将病毒插入Slide-A-Lyzer 10K透析盒。 清除卡带中的多余空气。
    6. 透析病毒在PBS + 10mM MgCl 2中至少4小时,伴随2次缓冲液更换(总共2L)或在4℃下在2L缓冲液中过夜。
    7. 使用注射器清除病毒。 等分并冷冻于-80℃

      图1.蔗糖梯度示意图。梯度负载(A)和离心后病毒带(B)的示意图。

  3. 星状病毒荧光聚焦测定
    1. 用2×10 4个Caco2细胞/孔接种96孔板。 生长3-4天,直到细胞达到100%汇合
    2. 用无菌PBS轻轻冲洗细胞2次。 胰酶消化和计数2-3孔,以计算细胞数/孔
    3. 加入100μlSF Caco2培养基(补充有0.3%BSA),并在37℃,5%CO 2下孵育1小时。
    4. 在含有0.3%BSA的SF Caco2培养基中制备10倍病毒稀释液
    5. 从细胞中除去培养基,并添加100微升的系列稀释液的细胞。 每个样品一式三份进行测定
    6. 在37℃,5%CO 2孵育1小时。 取出介质,并更换为含有0.3%BSA的SF Caco2介质
    7. 孵育板在37℃,5%CO 2,但不超过24小时至少10小时。
    8. 删除介质。 用PBS轻轻冲洗细胞3次。
    9. 在室温下固定在100μl的4%甲醛/PBS中20分钟
    10. 用〜100-200μlPBS冲洗3次。 所有冲洗均应使用此近似体积进行。
    11. 在室温下用100μl含有0.5%TritonX-100的PBS渗透10分钟
    12. 用PBS冲洗3次
    13. 在室温下在PBS中的100μl5%正常山羊血清中封闭1小时。 如果需要,板可以轻轻摇动
    14. 用PBS冲洗3次
    15. 与在PBS中的50μl1%正常山羊血清中的原代小鼠抗HAstV-1衣壳蛋白(8E7)1:100孵育在室温下1-2小时或在4℃过夜。 如果需要,板可以轻轻摇动
    16. 用PBS冲洗3次
    17. 与在PBS +1μg/ml DAPI中的50μl1%正常山羊血清中的第二山羊抗小鼠AlexaFluor 488 1:100孵育。 保持板远离此步骤的光。 如果需要,板可以轻轻摇动
    18. 用PBS冲洗3次。 向每个孔中加入约200μlPBS,并在荧光显微镜下观察(图2)
    19. 计算每ml的荧光聚焦单位(FFU/ml):%FITC +细胞x平均细胞数/孔x稀释因子= FFU/ml


      图2.星状病毒染色的实施例。未感染(左)和星状病毒感染(右)Caco2细胞的IFA染色。病毒衣壳显示为绿色,细胞核呈蓝色。

笔记

  1. 对于按照该方案制备的繁殖星状病毒原种,用MOI:1(如果可能)感染。 如果使用没有在猪胰蛋白酶存在下生长的粪便滤液或原液,在吸附到Caco2细胞上之前,用10μg/ml猪胰蛋白酶预温育滤液/原液1小时。
  2. 在90分钟感染期间,每15分钟摇动一次细胞
  3. 通过在处理之前在10%vol/vol漂白剂中孵育至少30分钟来灭活接种物中的病毒; 高压灭菌接种物; 或在1%vol/vol的Virkon S中孵育30分钟
  4. 病毒带的可视化可以通过在超速离心管后面放置黑色背景,或通过在罩/房间中关闭灯并在带上照射光来增加。

食谱

  1. Caco2细胞培养基 MEM补充:
    1%glutamax
    1%丙酮酸钠 10%热灭活的FBS
  2. 无血清(SF)Caco2培养基
    MEM补充:
    1%glutamax
    1%丙酮酸钠
  3. TN缓冲区
    50mM Tris(pH7.5) 100 mM NaCl
    灭菌

致谢

该协议改编自以前的出版物DuBois等人(2013); Moser和Schultz-Cherry(2008); 和Willcocks等人(1990)。 这项研究的资金由儿童感染防御中心,哈特韦尔基金会和美国黎巴嫩叙利亚联合慈善机构和圣犹达儿童研究医院提供。

参考文献

  1. DuBois,R.M.,Freiden,P.,Marvin,S.,Reddivari,M.,Heath,R.J.,White,S.W.and Schultz-Cherry,S。(2013)。 禽星状病毒衣壳尖峰的晶体结构 J Virol 87(14):7853-7863。
  2. Moser,L.A。和Schultz-Cherry,S。(2008)。 通过ERK1/2抑制剂抑制星状病毒复制。 J Virol 82(15):7475-7482。
  3. Willcocks,M.,Carter,M.,Laidler,F。和Madeley,C。(1990)。 人类粪便星状病毒在连续细胞系中的生长和表征 Arch Virol   113(1-2):73-81
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Marvin, S., Meliopoulos, V. and Schultz-Cherry, S. (2014). Human Astrovirus Propagation, Purification and Quantification. Bio-protocol 4(6): e1078. DOI: 10.21769/BioProtoc.1078.
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