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Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies   

Chie Aoki-UtsuboChie Aoki-Utsubo*Ming ChenMing Chen*Hak  HottaHak Hotta  (*contributed equally to this work)
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Original research article

A brief version of this protocol appeared in:
Scientific Reports
Nov 2017

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, Neutralizing activity, Viral particle, Antiviral substance, Antibody, Viral infectivity assay, Negative staining electron microscopic analysis

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.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.
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