Virucidal Efficacy Test (ASTM E1053 and ASTM E2197)
Standard Test Methods to
Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate,
Nonporous Environmental Surfaces.
CREM Co is capable of performing virucidal efficacy tests on a verity variety of viruses such as Adenovirus, Coronavirus, Murine norovirus, FCV, Rotavirus ….for submitting data to the U.S. Environmental Protection Agency (EPA) and Health Canada using ASTM standard methods such as E1053 and E2197.
The EPA accepts data generated using E1053, but Health Canada accepts data generated by either one of the two methods. The major differences between the two methods are:
(a) E2197 uses disks of brushed stainless steel as carriers to better represent the uneven nature of environmental surfaces in the field; disks of other types of materials (e.g., plastic) can also be used, if needed; E1053 uses glass Petri dishes (100 mm diam.) as carriers.
(b) In E2197, the volume of the microbial inoculum/carrier is only 10 µL, which conserves high-titered pools of test organisms; While the the volume of the microbial inoculum/carrier in E1053 is 200 µL.
(c) In E2197 50 µL of the test disinfectant is required to cover each disk; in contrast, E1053, uses 2 mL of the test formulation is to be placed over the dried virus inoculum on each carrier.
(d) In E2197, immediately at the end of the contact time each disk is placed in 1 mL of neutralizer/eluent for virus recovery; in E1053, 2 mL of the neutralizer/eluent is added to each dish.
(e) In E2197 the test substance is diluted 20-fold during the neutralization process while in ASTM E1053 the test substance is diluted just two-fold. Therefore, in E2197 the level of cytotoxicity is reduced considerably.
In both methods, CREM Co Labs measures virus infectivity using the plaque-forming units (PFU) instead of the most-probable number (MPN) method (based on tissue culture infective dose 50% or TCID50 calculations) to determine the levels of viable virus in control and test samples (Baer& Kehn-Hall, 2014). As the name itself suggests, the MPN method estimates the MPN of infectious units of a given virus. Such an estimate requires a larger number of replicates for better statistical confidence (LaBarre & Lowy, 2001). In contrast, PFU assays are a more precise way of measuring virus infectivity as it entails the counting of readily visible plaques (foci of damage to the host cell monolayer). Each plaque thus represents an actual viable or infectious virus particle in the sample being assayed. In other words, each plaque corresponds to a colony-forming unit (CFU) in bacterial viability assays (Cooper 1962).
The level of viable virus is then calculated by determining the number of countable plaques in each dilution, and the assay is based on a series of sample dilutions rather than an indirect estimation of virus infectivity as in the MPN method. Plaque assays can be performed using individual vessels with cell monolayers or cluster plates mainly with 12 to 24 wells. We prefer to use 12-well plates because each well in them provides a large enough surface area for plaque development and ease of plaque counting. The use of such plates with three wells/dilution better conserves high-titered virus pools which can be difficult and expensive to produce.
Another advantage of using cluster plates is that, at the end of the assay, cell monolayers in them can be chemically fixed and then stained with a dye (e.g., crystal violet) not only to make the plaques visible and easier to count but also enable the storage of the plates for any subsequent review or examination. The efficacy of the tested technology becomes visible by comparing the pictures of test plates and control plates.
Literature cited
Baer, A., Kehn-Hall, K. (2014). Viral concentration determination through plaque assays: Using traditional and novel overlay systems. J. Vis. Exp. 93, e52065, doi:10.3791/52065.
Bidawid S, Farber JM, Sattar SA, Hayward S. (2000). Heat inactivation of hepatitis A virus in dairy foods. J Food Prot.
Bidawid S, Malik N, Adegbunrin O, Sattar SA, Farber JM. (2003). A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. J Virol Methods.
Dulbecco R, Vogt M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 99:167-182.
LaBarre, D.A. Lowy, J.L. (2001). Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays. J Virol Methods 96: 107-126.
Cooper, P.D. (1962). The plaque assay of animal Viruses. Advances in Virus Research, 8; 319-378.