Virucidal activity testing

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. 

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. 

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). 

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. 

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. 

EN 1656, 2000 EN 1657, 2000 prEN 14204, 2001 prEN 14675 WI 216040 Quantitative suspension test for the evaluation of bactericidal activity Quantitative suspension test for the evaluation of fungicidal activity Quantitative suspension test for the evaluation of mycobactericidal activity Quantitative suspension test for the evaluation of virucidal activity Quantitative suspension test for the evaluation of sporicidal activity... 

De Logu et al. (2000) investigated the inactivation of HSV-1 and HSV-2 and the prevention of cell-to-cell virus spread by the EO of the Asteraceae Santolina insularis. The plaque-reduction assay showed an IC50 values of 0.88 p.g/mL for HSV-1 and 0.7 xg/mL for HSV-2, respectively, whereas another test on Vero cells showed a cytotoxic concentration (CC50) of 112 p.g/mL, which leads to a CC50/IC50 ratio of 127 for HSV-1 and 160 for HSV-2. These findings indicate that the antiviral effect of the EO was caused by direct virucidal effects. There was no antiviral activity detected in a postattachment assay. Dne to attachment assays it was shown that virus adsorption was not reduced. Additionally, the reduction of plaqne formation assay indicated that the EO reduced cell-to-cell transmission of both HSV-1 and HSV-2. 

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. 

Chrysophanic acid (l,8-dihydroxy-3-methylanthraquinone), isolated from the Australian Aboriginal medicinal plant Dianella longifolia, has been found to inhibit the replication of poliovirus types 2 and 3 (Picornaviridae) in vitro (Fig. 3.7 Semple et al. 2001). The compound inhibited an early stage in the viral replication cycle, but did not have an irreversible virucidal effect on poliovirus particles. Chrysophanic acid did not have significant antiviral activity against five other viruses which were tested Coxsackievirus types A21 and B4, human rhinovirus type 2 (Picornaviridae), and the enveloped viruses Ross River virus (Jogaviridae) and HS V-1 (Herpesviridae). 

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