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Influenza Virus-cell Fusion Inhibition Assay
流感病毒细胞融合抑制试验   

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Abstract

During viral infection to host cells, several viruses undergo the process of endocytosis and pH-dependent fusion. By fusion of viral membrane with host cellular membrane, the viral core invades to host cytoplasm. A part of monoclonal antibodies against viral membrane protein have potential to inhibit the viral fusion step. Here we describe in vitro influenza virus-cell fusion inhibition assay. The infected cells expressing viral membrane protein, such as hemagglutinin (HA), on cellular surface are incubated with monoclonal antibodies targeting viral membrane protein. Then the cells are incubated under low pH condition. If the antibody does not inhibit the fusion step, we can see multinucleated giant cells.

Keywords: Endocytosis(endocytosis), PH-dependent fusion(pH依赖的融合), Viral entry(病毒进入), Hemagglutinin(hemagglutinin), Influenza virus(流感病毒)

Materials and Reagents

  1. Monkey kidney cell line CV-1 cells
  2. MDCK-propagated Influenza B viruses (B/Florida/4/2006 and B/Malaysia/2506/2004) provided by the National Institute of Infectious Diseases, Japan
  3. Minimal Essential Medium (MEM) (Sigma-Aldrich, catalog number: M4655 )
  4. Fetal Bovine Serum (FBS) (MP Biomedicals, catalog number: 2917054 )
  5. Phosphate buffered saline without Ca2+ and Mg2+ (PBS)
  6. Dulbecco’s Modified Eagle’s Medium F12 (DMEMF12) (Life Technologies, catalog number: 11320-033 )
  7. Acetylated trypsin (Sigma-Aldrich, catalog number: T6763 )
  8. Giemsa stain solution (Wako Pure Chemical Industries, catalog number: 079-04391 )
  9. Methanol (Nacalai tesque, catalog number: 21915-93 )
  10. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9576 )
  11. Specific pathogen
  12. Monoclonal antibody
  13. MEM powder (Nisshin EM, catalog number: 05901 )
  14. HEPES (Sigma-Aldrich, catalog number: H3375 )
  15. MES (Wako Pure Chemical Industries, catalog number: 344-08351 )
  16. Fusion medium (see Recipes)

Equipment

  1. 96-well cell culture plate (Corning, catalog number: 3596 )
  2. CO2 incubator (37 °C, 5% CO2)
  3. Light microscope

Procedure

  1. (Day 1) CV-1 cells in 100 μl MEM including 10% FBS are passaged to 96-well plate at 2.5 x 105/ml and incubated in the CO2 incubator for 24 h.
  2. (Day 2) The cells are rinsed with 100 μl PBS once, adsorbed with 50 μl viruses in MEM at an MOI of 0.3 for 1 h in the CO2 incubator.
  3. The cells are rinsed with 100 μl PBS once, added 100 μl DMEMF12 including 0.2% BSA and incubated for 24 h in the CO2 incubator.
  4. (Day 3) The cells are rinsed with 100 μl MEM twice.
  5. The cells in 100 μl MEM including 2.5 μg/ml acetyrated trypsin are incubated in the CO2 incubator for 15 min.
  6. They are rinsed with 100 μl MEM twice.
  7. 50 μl MEM containing different concentration of monoclonal antibody (concentration is 100, 25, 6.25, 1.5, 0.4 and 0.1 μg/ml) is incubated with the cells in different wells for 30 min in the CO2 incubator.
  8. After aspiration of medium, 100 μl fusion medium with each of pH is added and then incubated for 2 min in the CO2 incubator.
  9. The cells are rinsed with 100 μl MEM twice.
  10. The cells are suspended with 100 μl DMEMF12 including 0.2% BSA and incubated in the CO2 incubator for 3 h.
  11. After aspiration of medium, the cells are fixed using 100 μl absolute methanol for 30 sec.
  12. After aspiration of methanol, the cells are stained with 100 μl diluted Giemsa staining solution (1 drop per ml dH2O) for 30 min at room temperature.
  13. After rinsing the cells on tap water, the cells are observed by microscope (Figure 1).


    Figure 1. Giemsa staining of CV-1 cells in fusion inhibition assay. Arrow heads show multinuclear giant cells.

Recipes

  1. Fusion medium
    Mix 2x MEM with 10 mM MES and 10 mM HEPES
    Adjust to pH 5.0, 5.5, 6.0, 6.5 and 7.0
    Mess up to 1x MEM
    Autoclaved

Acknowledgments

This work was supported in part by the Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development (JST/JICA, SATREPS) (http://www.jst.go.jp/global/kadai/h2011_thailand.html); and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science to MY (#23790660).

References

  1. Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.

简介

在病毒感染宿主细胞期间,几种病毒经历内吞作用和pH依赖性融合的过程。 通过病毒膜与宿主细胞膜的融合,病毒核心侵入宿主细胞质。 抗病毒膜蛋白的单克隆抗体的一部分具有抑制病毒融合步骤的潜力。 在这里我们描述了体外流感病毒 - 细胞融合抑制试验。 将表达细胞表面上的病毒膜蛋白(例如血凝素(HA))的感染细胞与靶向病毒膜蛋白的单克隆抗体温育。 然后将细胞在低pH条件下温育。 如果抗体不抑制融合步骤,我们可以看到多核巨细胞。

关键字:endocytosis, pH依赖的融合, 病毒进入, hemagglutinin, 流感病毒

材料和试剂

  1. 猴肾细胞系CV-1细胞
  2. 由日本国家传染病研究所提供的MDCK繁殖的乙型流感病毒(B/Florida/4/2006和B/Malaysia/2506/2004)
  3. 最小必需培养基(MEM)(Sigma-Aldrich,目录号:M4655)
  4. 胎牛血清(FBS)(MP Biomedicals,目录号:2917054)
  5. 不含Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)
  6. Dulbecco's Modified Eagle's Medium F12(DMEMF12)(Life Technologies,目录号:11320-033)
  7. 乙酰化胰蛋白酶(Sigma-Aldrich,目录号:T6763)
  8. 吉姆萨染色溶液(Wako Pure Chemical Industries,目录号:079-04391)
  9. 甲醇(Nacalai tesque,目录号:21915-93)
  10. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9576)
  11. 特定病原体
  12. 单克隆抗体
  13. MEM粉末(Nisshin EM,目录号:05901)
  14. HEPES(Sigma-Aldrich,目录号:H3375)
  15. MES(Wako Pure Chemical Industries,目录号:344-08351)
  16. Fusion介质(参见配方)

设备

  1. 96孔细胞培养板(Corning,目录号:3596)
  2. CO 2培养箱(37℃,5%CO 2)中培养。
  3. 光学显微镜

程序

  1. (第1天)将包含10%FBS的100μlMEM中的CV-1细胞以2.5×10 5/ml /孔传代至96-孔板,并在CO 2 - 孵育器24小时
  2. (第2天)将细胞用100μlPBS漂洗一次,在MO 2中在MO 2中用50μl病毒在MEM中吸附1小时,在CO 2培养箱中。
  3. 将细胞用100μlPBS漂洗一次,加入100μl包含0.2%BSA的DMEMF12,并在CO 2培养箱中孵育24小时。
  4. (第3天)将细胞用100μlMEM洗涤两次
  5. 将包含2.5μg/ml乙酰化胰蛋白酶的100μlMEM中的细胞在CO 2培养箱中温育15分钟。
  6. 它们用100μlMEM洗涤两次。
  7. 将含有不同浓度的单克隆抗体(浓度为100,25,6.25,1.5,0.4和0.1μg/ml)的50μlMEM与不同孔中的细胞在CO 2培养箱中孵育30分钟 。
  8. 吸去培养基后,加入100μl每种pH的融合培养基,然后在CO 2培养箱中孵育2分钟。
  9. 细胞用100μlMEM洗涤两次。
  10. 将细胞用100μl包含0.2%BSA的DMEMF12悬浮,并在CO 2培养箱中孵育3小时。
  11. 吸取培养基后,用100μl无水甲醇固定细胞30秒
  12. 吸入甲醇后,在室温下用100μl稀释的吉姆萨染色溶液(1ml/ml dH 2 O)将细胞染色30分钟。
  13. 在自来水上冲洗细胞后,通过显微镜观察细胞(图1)

    图1.融合抑制测定中CV-1细胞的Giemsa染色。箭头显示多核巨细胞。

食谱

  1. 融合介质
    将2x MEM与10mM MES和10mM HEPES混合 调整到pH 5.0,5.5,6.0,6.5和7.0
    混乱到1个MEM
    高压灭菌

致谢

这项工作得到日本科学技术机构/日本国际合作机构,可持续发展科学和技术研究伙伴关系(JST/JICA,SATREPS)的部分支持(http://www.jst.go.jp/global/kadai /h2011_thailand.html); 和日本科学促进会MY(#23790660)的青年科学家助学金(B)。

参考文献

  1. Yasugi,M.,Kubota-Koketsu,R.,Yamashita,A.,Kawashita,N.,Du,A.,Sasaki,T.,Nishimura,M.,Misaki,R.,Kuhara,M.,Boonsathorn,N 。,Fujiyama,K.,Okuno,Y.,Nakaya,T.and Ikuta,K.(2013)。 人类单克隆抗体广泛中和乙型流感病毒 PLoS Pathog < em> 9(2):e1003150。
  • 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: © 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. Yasugi, M. and Ikuta, K. (2013). Influenza Virus-cell Fusion Inhibition Assay. Bio-protocol 3(24): e1004. DOI: 10.21769/BioProtoc.1004.
  2. Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T. and Ikuta, K. (2013). Human monoclonal antibodies broadly neutralizing against influenza B virus. PLoS Pathog 9(2): e1003150.
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