流感病毒细胞融合抑制试验

Mayo Yasugi; Kazuyoshi  Ikuta

doi:10.21769/BioProtoc.1004

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Peer-reviewed

Influenza Virus-cell Fusion Inhibition Assay

流感病毒细胞融合抑制试验

Mayo Yasugi

Kazuyoshi Ikuta email

发布: 2013年12月20日第3卷第24期 DOI: 10.21769/BioProtoc.1004 浏览次数: 11343

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Feb 2013

Submit your protocol to the Special Issue on Technologies for RNA Detection

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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

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

Equipment

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

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Yasugi, M. and Ikuta, K. (2013). Influenza Virus-cell Fusion Inhibition Assay. Bio-protocol 3(24): e1004. DOI: 10.21769/BioProtoc.1004.
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.