搜索

Severe Fever with Thrombocytopenia Syndrome Virus Infection of Cell Cultures
高烧并带有细胞培养物血小板减少综合症病毒感染的研究   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Journal of Virology
Jan 2014

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) can infect multiple cells, such as Vero, RD, BHK21, HUVEC and 293T. Vero cells are highly susceptible, so they were used to isolate virus from sera of patients, as well as for producing virus in the laboratory. Other cells can be used for additional functional analysis.

Materials and Reagents

  1. Vero cells (acquired from ATCC, CCL-81 )
  2. SFTSV from patients sera
  3. Dulbecco’s modified Eagle medium (DMEM) + 4.5 g/L D-glucose with L-Glutamine (Life Technology, catalog number: C11965500BT )
  4. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10091148 )
  5. Penicillin streptomycin (PS) (Life Technologies, Gibco®, catalog number: 15140-122 )
  6. Antibody against nuclear protein (NP) of SFTSV
    Note: Produced in rabbit by the Antibody Facility of our institute (Sun et al., 2014), the antibody was affinity purified, 1 mg/ml, the working concentration is 5 μg/ml.
  7. TRIzol reagent (Life Technologies, InvitrogenTM, catalog number: 15596-026 )
  8. Reverse Transcriptase M-MLV (RNase H) [Takara Biotechnology (Dalian), catalog number: D2639A ]
  9. SYBR Premix Ex TaqTM Perfect Real Time [Takara Biotechnology (Dalian), catalog number: DRR039A ]
  10. Methanol
  11. 4',6-diamidino-2-phenylindole (DAPI)
  12. Alexa Fluor 488 goat anti-rabbit (Life Technologies, InvitrogenTM, catalog number: A11008 )
  13. Hipure Plasmid Filter Midiprep Kit (Life Technologies, catalog number: K210005 )
  14. Maintaining cell culture (see Recipes)
  15. Infection cell culture (see Recipes)
  16. Mounting media (see Recipes)

Equipment

  1. T25 flask (25 cm2) (BD, FalconTM, catalog number: 353108 )
  2. Tissue culture plate, 24-well flat bottom (BD, FalconTM, catalog number: 353047 ) or 48-well plate (BD, FalconTM, catalog number: 353078 )
  3. 25-cm bottles
  4. 37 °C, 5% CO2 incubator
  5. 7500 fast Real Time PCR System (Life Technologies, Applied Biosystems®)
  6. Zeiss LSM510 Meta laser scanning confocal microscope

Procedure

  1. Virus isolation
    1. Vero cells were seeded in T25 flasks with 80% confluence, cultured in 5 ml DMEM/10% FBS/PS one day earlier before being inoculated with SFTSV patient sera.
      1. The sera were acquired from the hospital. The vein blood was collected from patients through the clinical laboratory of the hospital and sera were acquired by centrifugation at 2,500 rpm.
      2. The virus in the sera of patients was first quantified by quantitive real time PCR with specific primers designed for amplifying NP of SFTSV (see below). Serum with high SFTSV concentration (about 108/ml) was used to isolate SFTSV.
      3. A total of 500 μl of the patient’s sera was inoculated in 4.5 ml DMEM/2% FBS in 25-cm bottles, incubated at 37 °C, 5% CO2 incubator, and changed with fresh DMEM/2% FBS in 24 h post infection.
    2. Cultured supernatant was collected 72 h after virus inoculation of Vero cells, and the supernatant was added to newly seeded Vero cells in a T25 flask Virus.
      1. All collected cultured supernatant was used for virus amplification for the first two to three passages, and then 2.5 ml supernatant was used to amplify the virus.
      2. The virus was amplified for 3 to 5 generations with cultured supernatant till the copies of NP reached 108 to 109/μl.
      3. SFTSV virus in DMEM/2% FBS/PS was distributed to 1.5 ml micro-centrifuge tubes and stored at -80 °C, avoiding freeze thaw cycles, even though the virus is not that sensitive to the free-thaw process.
    3. Titration of virus measured by quantitative Real Time-PCR.
      The supernatant was spun at 3,000 rpm for 5 min. 200 μl cultured supernatant was added to 1 ml TRIzol reagent.
      1. Total RNA was isolated from supernatant using TRIzol reagent according to the manufacturer’ instructions. 2 μg of total RNA was used to synthesize cDNA using Reverse Transcriptase M-MLV (RNase H) in 20 μl reaction following the user manual.
      2. Next, 2 μl cDNA from each sample was used for quantitive real-time PCR by using SYBR Premix Ex Taq TM Perfect Real Time according to the manufacturer’s instructions. Primers (FW: AGCAGCTCAATTTGACTGAG, RW: TCATCTCCACCTGTCTCCTT) were designed to amplify the 87 bp fragment of NP.
      3. The full length NP DNA was amplified from virus cDNA, and was cloned to pCI-neo Vector; the plasmid was prepared using Hipure Plasmid Filter Midiprep Kit according to the user manual.
      4. The concentration of the plasmid was quantified, and DNA copies were calculated according to their molecular mass. These were diluted to serial copies (101 to 107) for making the standard curve, after which the quantitative real time-PCR was performed.
        20 μl of PCR reaction include:
        10 μl 2x SYBR Premix ExTaq
        0.4 μl each of 10 μM Forward primer and Reverse primer
        0.4 μl ROX Reference Dye II
        6.8 μl H2O
        2 μl DNA sample
        Following PCR were performed at 95 °C 10 sec, 40 repeat cycles of 95 °C 5 sec and 60 °C 30 sec, and dissociation at last.
    4. The titration of the virus can also be determined by immunostaining with antibody against nuclear protein (NP) of SFTSV.
      Vero or HUVEC cells were seeded in 24 well plates with 80% confluence one day earlier and infected with different amounts of SFTSV collected after 5 generations (such as 2 μl, 5 μl, 10 μl, 20 μl to 40 μl in DMEM/2% FBS) for two hours in 37 °C, 5% CO2 incubator, washed with PBS for twice in 2 h post infection and change with DMEM/2% FBS.
      After one day (24 h) infection, the cells were washed with 500 μl PBS once, incubated for 2 to 5 min for every washing step with PBS, and then the cells were fixed with 500 μl 100% methanol for 10 min at RT, and then washed with 500 μl PBS once.
      Next, they were incubated with 200 μl antibodies against NP (produced in rabbit) at 5 μl/ml in PBS/2% BSA at 37 °C incubator for 1 h. Afterwards, the cells were washed with 500 μl PBS three times. The cells were incubated with 2 μg/ml of Alexa Fluor conjugated Goat anti-rabbit IgG at 37 °C incubator for 1 h and then washed with PBS three times. Following that, the cells were covered with 80 μl mounting medium. Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) in mounting media. Images were captured with fluorescence microscope at 10 or 20x lens. 

  2. Virus infection in vitro
    1. Vero or other cells were seeded one day earlier on 24 or 48 well plates depending on the experiment. Here, 48 well plates are used as an example.
    2. The 10 μl virus collected from the supernatant was diluted in 200 μl DMEM/2% FBS (MOI 1) and added to the cells.
    3. After the cells were incubated at the 37 °C, CO2 incubator for 2 h, the cell was washed with PBS once, and then changed with DMEM/2% FBS.
    4. In order to test the infection of SFTSV, after 14 to 24 h, the cell culture was collected, and total RNA was prepared using TRIzol reagent, and quantitive Real Time PCR were performed as above description. Alternatively, the cells were fixed with methanol and stained with NP antibody as above.

Representative data



Figure 1. Standard curve of quantitative real time-PCR for SFTSV NP at 102 to 106 copies. When quantitative PCR was performed, the plasmid NP- pCI-neo was diluted from 102 to 106. Next, 2 μl was added in 20 μl reaction using SYBR Premix Ex Taq as per the user manual, the reaction was duplicated, and PCR was performed on the 7500 fast Real Time PCR System. X axis is the quantity of the NP copies. The Y axis is the value of CT of PCR at different copies. Sample copies will be automatically generated by the program depending on the standard curve. The right figure is the amplification plot.


Figure 2. HUVEC cells were infected with SFTSV virus produced in Vero cells (vero-105 batch). Cells infected with different amounts of virus (2 μl, 5 μl, 10 μl, 20 μl and 40 μl) as indicated in the figure. Cells were stained with antibodies against NP (green), nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) in mounting media (blue). Images were acquired using a Zeiss LSM510 Meta laser scanning confocal microscope at 20 times.

Notes

  1. When SFTSV infects different cells, the detection time for RT-PCR or immune staining will be selected according the experimental requirement when multiple cells are used. This is because the cells have different sensitivities to SFTSV infection.

Recipes

  1. Maintaining cell culture
    DMEM/10% FBS/PS
  2. Infection cell culture
    DMEM/2% FBS/PS: 1 ml FBS, 500 μl PS and 48.5 ml DMEM for preparing 50 ml DMEM/2% FBS/PS
  3. Incubate condition
    37 °C 5% CO2 incubator
  4. Mounting media
    1:1 glycerol:PBS
    0.1% N-propyl gallate

Acknowledgments

We thank Zhaoling Sun from Yiyuan County Chinese Medical Hospital for providing us with sera samples from SFTSV patients. This work was supported by the Chinese Ministry of Science and Technology (2010CB530101, 2011CB812501) and the Beijing Municipal Government.

References

  1. Sun, Y., Qi, Y., Liu, C., Gao, W., Chen, P., Fu, L., Peng, B., Wang, H., Jing, Z., Zhong, G. and Li, W. (2014). Nonmuscle myosin heavy chain IIA is a critical factor contributing to the efficiency of early infection of severe fever with thrombocytopenia syndrome virus. J Virol 88(1): 237-248.

简介

血小板减少综合征病毒(SFTSV)的严重发热可以感染多种细胞,例如Vero,RD,BHK21,HUVEC和293T。 Vero细胞是高度易感的,因此它们用于从患者的血清中分离病毒,以及用于在实验室中产生病毒。 其他细胞可用于额外的功能分析。

材料和试剂

  1. Vero细胞(从ATCC获得,CCL-81)
  2. 来自患者血清的SFTSV
  3. Dulbecco改良的Eagle培养基(DMEM)+具有L-谷氨酰胺的4.5g/L D-葡萄糖(Life Technology,目录号:C11965500BT)
  4. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10091148)
  5. 青霉素链霉素(PS)(Life Technologies,Gibco ,目录号:15140-122)
  6. 抗SFTSV核蛋白(NP)抗体
    注意:由我们研究所的抗体设备在兔中产生(Sun 等人

    ,2014),将抗体亲和纯化,1mg/工作浓度为5μg/ml。
  7. TRIzol试剂(Life Technologies,Invitrogen TM ,目录号:15596-026)
  8. 反转录酶M-MLV(RNase H)[Takara Biotechnology(大连),目录号:D2639A]
  9. SYBR Premix Ex TaqTM完全实时[Takara Biotechnology(大连),目录号:DRR039A]
  10. 甲醇
  11. 4',6-二脒基-2-苯基吲哚(DAPI)
  12. Alexa Fluor 488山羊抗兔(Life Technologies,Invitrogen TM ,目录号:A11008)
  13. Hipure Plasmid Filter Midiprep Kit(Life Technologies,目录号:K210005)
  14. 维持细胞培养(参见配方)
  15. 感染细胞培养(见配方)
  16. 安装介质(参见配方)

设备

  1. T25烧瓶(25cm 2)(BD,Falcon TM,目录号:353108)。
  2. 组织培养板,24孔平底(BD,Falcon,目录号:353047)或48孔板(BD,FalconTM,目录号:353078)
  3. 25厘米的瓶子
  4. 37℃,5%CO 2培养箱
  5. 7500快速实时PCR系统(Life Technologies,Applied Biosystems )
  6. Zeiss LSM510 Meta激光扫描共聚焦显微镜

程序

  1. 病毒隔离
    1. 将Vero细胞接种在具有80%汇合的T25烧瓶中,在接种SFTSV患者血清之前一天在5ml DMEM/10%FBS/PS中培养。
      1. 血清从医院获得。 通过医院的临床实验室从患者收集静脉血,并通过以2500rpm离心获得血清。
      2. 首先通过使用设计用于扩增SFTSV的NP的特异性引物的定量实时PCR来定量患者血清中的病毒(参见下文)。 使用具有高SFTSV浓度(约10 8 s/ml)的血清来分离SFTSV。
      3. 将总共​​500μl的患者血清接种在25-cm瓶中的4.5ml DMEM/2%FBS中,在37℃,5%CO 2培养箱中孵育,并且用新鲜的DMEM/2%FBS。感染后24小时
    2. 在病毒接种Vero细胞72小时后收集培养的上清液,并将上清液加入到T25瓶病毒中新接种的Vero细胞中。
      1. 所有收集的培养上清液用于前两至三代的病毒扩增,然后使用2.5ml上清液扩增病毒。
      2. 将病毒用培养的上清液扩增3至5代,直到NP的拷贝达到10 8到10 10/sup /μl。
      3. 将在DMEM/2%FBS/PS中的SFTSV病毒分配到1.5ml微量离心管中并储存在-80℃下,避免冻融循环,即使病毒对自由解冻过程不敏感。 >
    3. 通过定量实时PCR测量的病毒滴定 将上清液以3,000rpm离心5分钟。将200μl培养上清液加入1ml TRIzol试剂中。
      1. 使用TRIzol试剂根据制造商的说明书从上清液中分离总RNA。使用逆转录酶M-MLV(RNase H)按照用户手册在20μl反应中使用2μg总RNA合成cDNA。
      2. 接下来,根据制造商的说明书,使用来自每个样品的2μlcDNA用于使用SYBR Premix Ex Taq TM Perfect Real Time的定量实时PCR。设计引物(FW:AGCAGCTCAATTTGACTGAG,RW:TCATCTCCACCTGTCTCCTT)以扩增NP的87bp片段。
      3. 从病毒cDNA扩增全长NP DNA,并克隆到pCI-neo Vector;根据用户手册使用Hipure Plasmid Filter Midiprep Kit制备质粒
      4. 定量质粒的浓度,并根据它们的分子量计算DNA拷贝。将它们稀释成系列拷贝(10×10至10×10 7) 作为标准曲线,之后进行定量实时PCR。
        20μlPCR反应包括:
        10μl2x SYBR Premix ExTaq
        10μM正向引物和反向引物各0.4μl 0.4μlROX Reference Dye II
        6.8μlH 2 O 2/b 2μlDNA样品
        在95℃10秒,95℃5秒和60℃30秒的40个重复循环进行PCR后,最终解离。
    4. 病毒的滴定也可以通过用针对SFTSV的核蛋白(NP)的抗体进行免疫染色来测定。
      将Vero或HUVEC细胞接种在24孔板中,一天前80%汇合,并感染5代后收集的不同量的SFTSV(例如2μl,5μl,10μl,20μl至40μl在DMEM/2% FBS)在37℃,5%CO 2培养箱中孵育2小时,在感染后2小时用PBS洗涤2次,并用DMEM/2%FBS改变。
      一天(24小时)感染后,用500μlPBS洗涤细胞一次,每次用PBS洗涤步骤孵育2-5分钟,然后将细胞用500μl100%甲醇在室温固定10分钟,然后用500μlPBS洗涤一次。
      接下来,将它们与200μl针对NP(在兔中产生)的抗体以5μl/ml在PBS/2%BSA中在37℃培养箱中孵育1小时。然后,用500μlPBS洗涤细胞三次。将细胞与2μg/ml Alexa Fluor缀合的山羊抗兔IgG在37℃孵育箱孵育1小时,然后用PBS洗涤3次。然后,用80μl封固剂包被细胞。使用4',6-二脒基-2- 苯基吲哚(DAPI)。 用荧光显微镜在10或20x透镜下捕获图像。

  2. 病毒感染体外
    1. 根据实验,将Vero或其它细胞在一天前接种在24或48孔板上。 这里,使用48孔板作为实例
    2. 将从上清液收集的10μl病毒在200μlDMEM/2%FBS(MOI 1)中稀释并加入细胞中。
    3. 将细胞在37℃,CO 2培养箱中孵育2小时后,用PBS洗涤细胞一次,然后用DMEM/2%FBS改变。
    4. 为了测试SFTSV的感染,在14至24小时后,收集细胞培养物,使用TRIzol试剂制备总RNA,并如上所述进行定量实时PCR。 或者,将细胞用甲醇固定并用如上所述的NP抗体染色

代表数据



图1.在10° 2 至10 的SFTSV NP的定量实时PCR的标准曲线。 。当进行定量PCR时,将质粒NP-pCI-neo从10稀释至10 6 。接下来,使用SYBR Premix Ex Taq按照用户手册在20μl反应中加入2μl,重复反应,并在7500快速实时PCR系统上进行PCR。 X轴是NP拷贝的数量。 Y轴是不同拷贝的PCR的CT值。样品拷贝将由程序根据标准曲线自动生成。右图是扩增图。


图2.用在Vero细胞(vero-105批次)中产生的SFTSV病毒感染HUVEC细胞。用不同量的病毒(2μl,5μl,10μl,20μl和40μl)感染的细胞),如图所示。用针对NP(绿色)的抗体对细胞染色,在封固介质(蓝色)中使用4',6-二脒基-2-苯基吲哚(DAPI)对核进行染色。使用Zeiss LSM510 Meta激光扫描共聚焦显微镜获得图像20次。

笔记

  1. 当SFTSV感染不同的细胞时,当使用多个细胞时,根据实验需要选择用于RT-PCR或免疫染色的检测时间。 这是因为细胞对SFTSV感染具有不同的敏感性。

食谱

  1. 维持细胞培养
    DMEM/10%FBS/PS
  2. 感染细胞培养
    DMEM/2%FBS/PS:1ml FBS,500μlPS和48.5ml DMEM,用于制备50ml DMEM/2%FBS/PS。
  3. 孵育条件
    37℃5%CO 2培养箱
  4. 安装介质
    1:1甘油:PBS
    0.1%的没食子酸丙酯

致谢

我们感谢益源县中医医院的赵浩红为我们提供了来自SFTSV患者的血清样本。 这项工作得到了中国科学技术部(2010CB530101,2011CB812501)和北京市政府的支持。

参考文献

  1. Sun,Y.,Qi,Y.,Liu,C.,Gao,W.,Chen,P.,Fu,L.,Peng,B.,Wang,H.,Jing,Z.,Zhong, 李,W.(2014)。 非肌肉肌球蛋白重链IIA是促进早期感染伴有血小板减少症的严重发热的效率的关键因素 综合征病毒。 J Virol 88(1):237-248。
  • 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: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sun, Y. and Li, W. (2014). Severe Fever with Thrombocytopenia Syndrome Virus Infection of Cell Cultures. Bio-protocol 4(20): e1274. DOI: 10.21769/BioProtoc.1274.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。