抗病毒物质和抗体的杀灭病毒活性及中和活性检测

Chie Aoki-Utsubo; Ming Chen; Hak  Hotta

doi:10.21769/BioProtoc.2855

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Peer-reviewed

Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies

抗病毒物质和抗体的杀灭病毒活性及中和活性检测

CA Chie Aoki-Utsubo ^*

MC Ming Chen ^*

Hak Hotta email

(*contributed equally to this work) 发布: 2018年05月20日第8卷第10期 DOI: 10.21769/BioProtoc.2855 浏览次数: 12513

评审: Vamseedhar RayaproluBalaji Olety AmaranathKathrin Sutter

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Abstract

In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of the viral particles. The viral infectivity can be measured in cell culture system by means of plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID₅₀) assay, etc. Morphologically, disruption of viral particles can be demonstrated by negative staining electron microscopic analysis of viral particles. In this article, we describe methods to assess virucidal activity in a broad definition.

Keywords: Virucidal activity (杀病毒活性)

Neutralizing activity (中和活性)

Viral particle (病毒颗粒)

Antiviral substance (抗病毒物质)

Antibody (抗体)

Viral infectivity assay (病毒感染力测定)

Negative staining electron microscopic analysis (负染色电镜分析)

Background

Viruses are small intracellular parasites that hijack host cell machinery to replicate their own genome. At the initial step of the viral life cycle, infectious viral particles attach (bind) to particular host proteins, called viral receptors, on the surface of the target cells, followed by viral penetration (internalization and/or fusion) into intracellular compartments of the host cells, where the subsequent steps of the viral life cycle proceed to produce progeny virions (Scheel and Rice, 2013).

Virucidal activity in a narrow definition represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to interact with and functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of viral particles, as in the case of antibody-mediated neutralization.

We have recently reported that an isoform of secreted phospholipase A₂ obtained from snake venom (Chen et al., 2017) and a peptide from scorpion venom (El-Bitar et al., 2015) possess strong virucidal activity against viruses that belong to the family Flaviviridae by targeting the lipid bilayer of the viral envelope, which is acquired from the endoplasmic reticulum membrane of the host cells. It was also reported that one of the host defense peptides from the skin of the South Indian frog has a strong virucidal activity against H1 hemagglutinin-bearing human influenza virus by targeting the conserved stalk of H1 hemagglutinin (Holthausen et al., 2017). In this article, we describe a number of useful methods by which to measure virucidal activity in a broad definition, such as plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID₅₀) assay and negative staining electron microscopic analysis.

Materials and Reagents

Disposable tips
1. 10 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3510-05 )
2. 200 μl capacity (Thermo Fisher Scientific, Molecular BioProducts, catalog number: 3900 )
3. 1 ml capacity (FUKAEKASEI and WATSON, catalog number: 110-502C )
100 mm culture dish (Corning, Falcon^®, catalog number: 353003 )
6-well culture plate (Corning, Falcon^®, catalog number: 353046 )
12-well culture plate (Corning, Falcon^®, catalog number: 353043 )
24-well culture plate (Corning, Falcon^®, catalog number: 353047 )
96-well culture plate (Corning, Falcon^®, catalog number: 353072 )
1.5 ml microcentrifuge tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
15 ml tube (Corning, Falcon^®, catalog number: 352196 )
Cover slip (13 x 13 mm; Matsunami Glass, catalog number: C013001 )
Microscope slide (Matsunami Glass, catalog number: S2215 )
Disposable serological pipette
1. 1 ml capacity (Corning, Falcon^®, catalog number: 356521 )
2. 5 ml capacity (IWAKI, catalog number: 7153-005 )
3. 10 ml capacity (IWAKI, catalog number: 7154-010 )
Filter paper (ATTO, catalog number: CB-06A-20A )
Viruses (Chen et al., 2017):
1. Hepatitis C virus (HCV, J6/JFH-1 strain)
2. Dengue virus (DENV, Trinidad 1751 strain)
3. Japanese encephalitis virus (JEV, Nakayama strain)
4. Influenza A virus (FLUAV, A/Udorn/307/72[H3N2])
5. Sendai virus (SeV, Fushimi strain)
6. Herpes simplex virus type 1 (HSV-1, CHR3 strain)
7. Coxsackievirus B3 (CV-B3, Nancy strain)
8. Vesicular stomatitis New Jersey virus (VSNJV)
9. Sindbis virus (SINV)
10. Encephalomyocarditis virus (EMCV, DK-27 strain)
Huh7it-1 cells (Apriyanto et al., 2016)
Note: Huh7it-1 cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Vero cells (ATCC, catalog number: CCL-81 )
Note: Vero cells are susceptible to all viruses described above (HCV, DENV, JEV, FLUAV, SeV, HSV-1, CV-B3, VSNJV, SINV and EMCV).
Antibodies
1. Rabbit polyclonal antibody against DENV PrM (Gene Tex, catalog number: GTX128093 )
2. Mouse monoclonal antibody against DENV type 2 (3H5; Hotta et al., 1984)
3. UV-inactivated anti-HCV human serum (Bungyoku et al., 2009)
4. Anti-HCV E2 neutralizing antibody #55 (Shimizu et al., 2013)
5. Rabbit antiserum against CV-B3 (DENKA SEIKEN, catalog number: 300638 )
6. Rabbit antiserum against FLUAV (Shimizu et al., 1985)
7. Rabbit antiserum against SeV (Hayashi et al., 1991)
8. Rabbit antiserum against HSV-1 (Hayashi et al., 1986)
9. FITC-conjugated goat anti-human IgG (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 104AG )
10. Alexa Flour488-conjugated goat anti-mouse IgG (Thermo Fisher Science, catalog number: A-11001 )
11. Alexa Flour488-conjugated goat anti-rabbit IgG (Thermo Fisher Science, catalog number: A-11008 )
High glucose Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, catalog number: 044-29765 )
Phospholipase A₂ from Naja mossambica snake venom (Sigma-Aldrich, catalog number: P7778 ) (Chen et al., 2017)
Trypsin-EDTA solution (Wako Pure Chemical Industries, catalog number: 209-16941 )
Crystal violet (Wako Pure Chemical Industries, catalog number: 038-04862 )
MEM with non-essential amino acids (Thermo Fisher Science, Gibco^TM, catalog number: 10370021 )
Fetal bovine serum (FBS; Biowest, catalog number: S1820 )
Penicillin-Streptomycin solution (Wako Pure Chemical Industries, catalog number: 168-23191 )
Methyl Cellulose 4000 (Wako Pure Chemical Industries, catalog number: 136-02155 )
4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries, catalog number: 163-20145 )
Formaldehyde solution (AppliChem, catalog number: A3592,0500 )
Gram Hacker’s Stain Solution I (MUTO PURE CHEMICALS, catalog number: 41162 )
Triton X-100 (Wako Pure Chemical Industries, catalog number: 169-21105 )
Bovine serum albumin (BSA; Wako Pure Chemical Industries, catalog number: 015-21274 )
Hoechst 33342 solution (Thermo Fisher Scientific, Molecular Probes, catalog number: H3570 )
Vectashield mounting solution (Vector Laboratories, catalog number: H-1000 )
Formvar-coated nickel grid (Electron Microscopy Sciences, catalog number: FF200-Ni )
2% phosphotungstic acid (Wako Pure Chemical Industries, catalog number: 582-66852 )
Sodium chloride (NaCl; Wako Pure Chemical Industries, catalog number: 191-01665 )
Potassium chloride (KCl; Wako Pure Chemical Industries, catalog number: 163-03545 )
Disodium Hydrogen Phosphate (Na₂HPO₄·12H₂O, NACALAI TESQUE, catalog number: 31722-45 )
Potassium phosphate monobasic (KH₂PO₄; Wako Pure Chemical Industries, catalog number: 169-04245 )
Complete medium for cell culture (see Recipes)
10x phosphate-buffered saline (PBS[-]) (see Recipes)
Overlay medium (see Recipes)

Equipment

Micropipette (Gilson, P20, P200, P1000)
1. P20 (Gilson, catalog number: F123600 )
2. P200 (Gilson, catalog number: F123601 )
3. P1000 (Gilson, catalog number: F123602 )
Multichannel micropipette (10-100 μl) (Eppendorf, catalog number: 3125000036 )
Hemocytometer chamber (e.g., Erma, catalog number: 03-303-1 )
Biosafety cabinet (e.g., PHC, model: MHE-S1301A2 )
CO₂ incubator (e.g., PHC, model: MCO-20AIC )
Autoclave (e.g., TOMY DIGITAL BIOLOGY, model: SX-500 )
Refrigerated tabletop centrifuge (e.g., Eppendorf, model: Centrifuge 5424 )
Vortex (e.g., Scientific Industries, model: Vortex-Genie 2 )
-80 °C freezer (e.g., PHC, model: MDF-384 )
Inverted microscope (e.g., Olympus, model: CKX53 )
Fluorescent microscope (e.g., ZEISS, model: Axio Vert. A1 )
Multilabel Plate Counter (PerkinElmer, model: 1420 ALBOSX )
Transmission electron microscope (Hitachi, model: HT7700 TEM )

Procedure

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Aoki-Utsubo, C., Chen, M. and Hotta, H. (2018). Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies. Bio-protocol 8(10): e2855. DOI: 10.21769/BioProtoc.2855.