Evaluation of Antiseptic Antiviral Activity of Chemical Agents

Chloé Geller1, Chantal Finance1, Raphaël Emmanuel Duval1

1 Nancy‐University, CNRS, Nancy Cedex, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.10
DOI:  10.1002/0471143030.cb2610s51
Online Posting Date:  June, 2011
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Abstract

Antiviral antisepsis and disinfection are crucial for preventing the environmental spread of viral infections. Emerging viruses and associated diseases, as well as nosocomial viral infections, have become a real issue in medical fields, and there are very few efficient and specific treatments available to fight most of these infections. Another issue is the potential environmental resistance and spread of viral particles. Therefore, it is essential to properly evaluate the efficacy of antiseptics‐disinfectants (ATS‐D) on viruses. ATS‐D antiviral activity is evaluated by (1) combining viruses and test product for an appropriately defined and precise contact time, (2) neutralizing product activity, and (3) estimating the loss of viral infectivity. A germicide can be considered to have an efficient ATS‐D antiviral activity if it induces a >3 or >4 log10 reduction (American and European regulatory agency requirements, respectively) in viral titers in a defined contact time. This unit describes a global methodology for evaluating chemical ATS‐D antiviral activity. Curr. Protoc. Cell Biol. 51:26.10.1‐26.10.22. © 2011 by John Wiley & Sons, Inc.

Keywords: antiseptic; antiviral; chemical agents; neutralization; gel‐filtration

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Quantitative Suspension Test for Antiviral ATS‐D Activity Evaluation Using Sephadex Gel‐Filtration Neutralization
  • Support Protocol 1: L‐132 Cell Culture
  • Support Protocol 2: Viral Culture and Titration by the End Point Dilution Method
  • Support Protocol 3: Cytotoxicity Assays
  • Support Protocol 4: Evaluation of Retention Rates by Sephadex Columns and UV‐Visible Spectrophotometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative Suspension Test for Antiviral ATS‐D Activity Evaluation Using Sephadex Gel‐Filtration Neutralization

  Materials
  • Combed cotton (e.g., Dutscher Scientific cat. no. 030232)
  • Test product (cytotoxicity determined in preliminary assays using protocol 4)
  • G‐10 or G‐25 Sephadex suspension (see recipe)
  • Deionized, sterile water, pH 7.0 (see important note below)
  • 7.5% (w/v) sodium bicarbonate (if required)
  • 1‐ml frozen viral suspension ( protocol 3)
  • Nine 96‐wells plates with confluent monolayer of L‐132 cells: seeded 48 hr before performing the assay ( protocol 2, plates for viral titration)
  • Bleach solution with 9.6% (w/v) active chlorine: 250 ml bleach diluted in 2 liters H 2O
  • MEM‐2: minimum essential medium with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), supplemented with 2% (v/v) fetal bovine serum (FBS, Invitrogen cat. no. 10270098)
  • 1 ml‐syringes
  • 1.5‐ml microcentrifuge tubes
  • Stainless steel scissors, sterile
  • Stainless steel dissecting forceps, sterile
  • Container for sterilizing column components (e.g., jar with lid)
  • 50‐ml conical centrifuge tubes with tops, sterile
  • 0.22‐µm syringe‐driven filter unit
  • 200× inverted microscope
  • 96‐well cell culture plates
  • 8‐channel multichannel pipettor and reagent reservoirs
  • Humidified, 37°C, 5% CO 2 cell culture incubator
  • Additional reagents and equipment for evaluating viral titers with using the endpoint dilution method ( protocol 3)
IMPORTANT NOTE: The quality of water (e.g., hardness due to the presence of ions like chloride, magnesium, or calcium) used in making the test solutions is very important because it can influence product activity. Therefore, deionized water is typically used. In addition, as autoclaving can acidify the water, the pH must be checked after autoclaving and the water neutralized, if necessary, by the addition of 7.5% (w/v) sodium bicarbonate (NaHCO 3). Hard water can be used, but if it is, the hardness should be precisely measured and specified. Indeed, hard water is recommended by some standards because it is more representative of conditions in the actual use of the product. However, deionized water allows easier assay standardization. Furthermore, autoclaving can induce an acidification of the water, so the pH must be checked. Indeed, pH‐induced variability in virus infectivity has been demonstrated with coronaviruses (Sturman et al., ).

Support Protocol 1: L‐132 Cell Culture

  Materials
  • L‐132 cells (ATCC #CCL‐5)
  • MEM‐5: with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), supplemented with 5% (v/v) fetal bovine serum (FBS, Invitrogen cat. no. 10270098)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA (Invitrogen cat.no.25300‐059)
  • FBS
  • Dimethyl sulfoxide (DMSO)
  • Isopropanol
  • Liquid nitrogen
  • MEM‐10: MEM with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen cat. no. 10270098)
  • MEM: MEM with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), without serum
  • 4% (v/v) trypan blue
  • 25‐cm2 and 75‐cm2 tissue culture flasks
  • Humidified, 37°C, 5% CO 2 cell culture incubator
  • 200× inverted microscope
  • 2‐ml cryotubes (Sarstedt cat.no.72.699.406)
  • Freezing container (e.g., by Nalgene, Thermo Scientific cat. no. 5100‐0001)
  • Hemacytometer
  • 96‐well plates (Sarstedt cat.no.83.1835.300)
  • 50‐ml test to be, sterile

Support Protocol 2: Viral Culture and Titration by the End Point Dilution Method

  Materials
  • 75 cm2‐flasks with confluent monolayers of L‐132 cells (two flasks; 48 hr incubation; protocol 2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 ml frozen virus suspension (HCoV 229E, ATCC #VR 740), ∼106 infectious particles/ml
  • MEM: minimum essential medium with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), serum‐free
  • MEM‐2: MEM, supplemented with 2% (v/v) fetal bovine serum (FBS, Invitrogen cat. no. 10270098)
  • 96‐well plates of confluent L‐132 cell monolayers seeded for viral titration ( protocol 2)
  • Bleach solution with 9.6% (w/v) active chlorine: 250 ml bleach diluted in 2 liters H 2O
  • May‐Grünwald solution (Merck cat. no.101424)
  • Giemsa solution (Merck cat. no.109204)
  • 33°C, 5% CO 2 cell culture incubator
  • 2‐ml cryovials
  • 8‐channel multichannel pipettor

Support Protocol 3: Cytotoxicity Assays

  Materials
  • Test product
  • Sterile, deionized water, pH 7.0
  • 96‐wells plates (four for each test) with 40% confluent L‐132 cells: seeded 48 hr before performing the assay ( protocol 2, plates for cytotoxicity testing)
  • MEM‐5: minimum essential medium with Glutamax and Earle's salts (Invitrogen cat. no. 41090‐093), supplemented with 5% (v/v) fetal bovine serum (FBS, Invitrogen cat. no. 10270098)
  • Bleach solution with 9.6% (w/v) active chlorine
  • Phosphate‐buffered saline (PBS: appendix 2A)
  • MTT (methyl thiazol tetrazolium) solution (see recipe)
  • SDS (sodium dodecyl sulfate) solution (see recipe)
  • NR solution (see recipe)
  • Solution A (see recipe)
  • Solution B (see recipe)
  • 0.22‐µm filter
  • 8‐channel multichannel pipettor
  • Scanning multiwell spectrophotometer
  • Additional reagents and equipment for preparing and using in‐house Sephadex columns ( protocol 1)

Support Protocol 4: Evaluation of Retention Rates by Sephadex Columns and UV‐Visible Spectrophotometry

  Materials
  • Test product
  • Sterile, deionized water, pH 7.0
  • UV‐visible spectrophotometer
  • Additional reagents and equipment for preparing and using in‐house Sephadex columns ( protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   AFNOR (Agence Française de NORmalisation). 2007. Essai virucide quantitatif de suspension pour les antiseptiques et désinfectants chimiques utilisés en médecine humaine ‐ Méthode d'essai et prescriptions (phase 2, étape 1). NF EN 14476+A1. http://www.afnor.org.
   ASTM (American Society for Testing and Materials). 2004. Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations. E1482‐04.
   Borenfreund, E. and Puerner, J.A. 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24:119‐124.
   Bradburne, A.F. 1972. An investigation of the replication of coronaviruses in suspension cultures of L132 cells. Arch. Gesamte Virusforsch. 37:297‐307.
   Geller, C., Fontanay, S., Finance, C., and Duval, R.E. 2009. A new Sephadex‐based method for removing microbiocidal and cytotoxic residues when testing antiseptics against viruses: Experiments with a human coronavirus as a model. J. Virol. Methods 159:217‐226.
   Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55‐63.
   Reed, L.J. and Muench, H. 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27:493‐497.
   Sturman, L.S., Ricard, C.S., and Holmes, K.V. 1990. Conformational change of the coronavirus peplomer glycoprotein at pH 8.0 and 37°C correlates with virus aggregation and virus‐induced cell fusion. J. Virol. 64:3042‐3050.
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