Virucidal tests

An evaluation of antiviral properties of various EOs from South American plants was carried out by Duschatzky et al. (2005). The authors assessed the cytotoxicity and in vitro inhibitory activity of the EOs against HSV-1, DENV-2, and JUNV by a virucidal test. The best results were observed with the EOs of Heterothalamus alienus (Asteraceae) and Buddleja cordobensis (Scrophulariaceae) against JUNV, with virucidal concentration 50% (VC50) values of 44.2 and 39.0 ppm and therapeutic indices (cytotoxicity to virucidal action ratio) of 3.3 and 4.0, respectively. The oils caused the inhibitory effect interacting directly with the virions. 

In the United States, several tests using bacteria as the test organisms have been adopted as standardized methods. The primary test used is the use-dilution method from the AOAC (Association of Official Analytical Chemists). The American Society for Testing and Materials (ASTM) has also developed virucidal test procedures. In western Europe, disinfection tests are published as national standards in France (AFNOR), the United Kingdom (BSI), and Germany (DIN). 

The testing of disinfectants for virucidal activity is not an easy matter. As pointed out earlier (Chapter 3), viruses are unable to grow in artificial culture media and thus some other system, usually employing living cells, must be considered. One such example is tissue culture, but not all virus types can propagate under such circumstances and so an alternative approach has to be adopted in specific instances. The principles of such methods are given below. 

For assaying herpes virus, monolayers of baby hamster kidney (BHK) cells are used. Virus titre is expressed as the number of plaque-forming units (pfu) per millilitre before and after exposure to a disinfectant, so that the virucidal efficacy of the test agent can be determined. A diagrammatic representation is given in Fig. 11.7. 

Tyler R. Ayliffe G.A.J. (1987) A surface test for virucidal activity of disinfectants preliminary study with herpes virus. J Hasp Infect, 9, 22-29. 

The purpose of this study was to evaluate the antiviral properties of the inventive silver colloids (10 ppm and 32 ppm) against Influenza A (HINT) virus or Avian Influenza A (H3N2) virus ( bird flu ) when exposed (in suspension) for a specified exposure period(s). The protocol used is a modification of the Standard Test Method for Efficacy of Virucidal Agents Intended for Special Applications (ASTM E1052). 

The test medium used for the virucidal assays was Minimum Essential Medium (MEM) supplemented with 1-10% (v/v) heat inactivated FBS. The medium may also be supplemented with one or more of the following 10 pg/ml gentamicin, 100 units/ml penicillin, and 2.5 pg/ml amphotericin B. 

Each cytotoxicity control mixture (above) was challenged with low titer stock virus to determine the dilution of test substance at which virucidal activity, if any, was retained. Dilutions that showed virucidal activity will not be considered in determining reduction of the virus by the test substance. 

As previously described, 0.1 ml of each test and control parameter following the exposure period was added to a 0.9 ml aliquot of neutralizer followed immediately by 10-fold serial dilutions in test medium to stop the action of the test substance. To determine if the neutralizer chosen for the assay was effective in diminishing the virucidal activity of the test substance, low titer stock virus was added to each dilution of the test substance-neutralizer mixture. This mixture was assayed for the presence of virus (neutralization control above). 

Gaustad, J.W., McDuff, C.R., and Hatcher, H.J., 1974. Test method for the evaluation of virucidal efficacy of three common liquid surface disinfectants on a simulated environmental surface. Appl. Microbiol., 28 748-752. 

Efficacy tests have demonstrated that NP 5.5 HW is an effective bactericide, fungicide, and virucide in hard water up to 400 ppm hardness (as CaC03) in the presence of organic soil (5% blood serum). ... 

Even though tap water may represent a stronger challenge, its quality, as well as the nature and levels of disinfectants in it, varies both temporarily and geographically. In view of this, water with a standard level of hardness in it (e.g., 200-400 ppm CaCOs) makes for a more desirable diluent in tests for virucidal activity. 

For accurate and meaningful results, the virucidal activity of the test formulation must be arrested immediately and effectively at the end of the contact time without the process itself killing the virus or enhancing cytotoxicity. This can be achieved by either the addition of a neutralizer or by dilution of the virus-germicide mixture, or by a combination of both [31,32]. Whichever approach is adopted, its effectiveness must be properly validated before the test results can be accepted. 

Any good test for vimcidal activity must incorporate a measurement of infectivity of the challenge vims. This is essential to determine the level of loss in viral infectivity for the host system used. In many tests for virucidal activity, only very small fractions (often only 1-10%) of the test sample eluates are titrated for infectious vims, and the absence of infectivity in the host system may be taken as evidence of the formulation s effectiveness. The limit of detection for the assay system is rarely quoted, but when no infectious virus is detected, the results should be quoted as less than the theoretical detection limit. For a higher level of confidence in the results, it is desirable to titrate most or all of the test sample eluates. In order to save time and materials, ultrafiltration can be used to reduce the volume of the eluates using filters that allow very little protein binding. 

CEN Virucidal Suspension Test for Chemical Disinfectants and Antiseptics Used in Human Medicine DRAFT A suspension of the test virus, with or without a soil load, is mixed with the test agent and the mixture held at 20°C for the desired contact time. The mixture is then titrated for infectious virus. Under review by CEN/TC 216... 

In carrier tests to determine the virucidal activity of other types of chemical germicides, there is no provision to include any virus loss due to precleaning of an object or its posttreatment rinsing. In the case of semi-critical medical devices,... 

SA Sattar, VS Springthorpe. Methods for testing the virucidal activity of chemicals. In SS Block, ed. Disinfection, Sterilization, and Preservation. New York Lippincott Williams Wilkins, 2001, pp. 1391-1412. 

ASTM E1053-97, Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Environmental Surfaces, ATSM International, West Conshohocken, PA, 2002. 

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