Rhinovirus Virucidal Assay Final Report

ANTIMICROBIAL ACTIVITY OF ACS 200 SILVER SOLUTION Using Rhinovirus 14 (RV, strain 1059)
Test Solution: ACS 200
February 06, 2015

PREPARED FOR:
Results RNA, LLC
BY:
Dale Barnard, Ph.D.

Department of Microbiology, Virology
Utah State University

Sponsor: Results RNA

Test start date: 06 Feb 2015

Test Compound: ACS 200

Viruses: Cells:
Rhinovirus 14 (RV, strain1059) HeLa-Ohio1

Cell Growth Medium: Minimal Essential Medium (MEM) with 5% Fetal bovine serum
Virucidal Test medium: MEM
Virus Propagation medium:
CHIKV, MERS-CoV, WNV: MEM + 2% FBS + 0.22% NaHCO3 + 50 µg/ml Gentamicin
IVA: MEM 1X + 1 mM EDTA + 0.25%Trypsin
RV: MEM + 2% FBS + 25mM MgCl + 50 µg/ml Gentamicin

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Virucidal Procedure

The virucidal assay is to determine if virus is inactivated by direct contact with the test compound. Compound was diluted as needed in purified water. Equal amounts of test compound and virus were mixed in a microtube and allowed to incubate for 1 h at 37ºC. Surviving virus was then titered using standard methods on the appropriate cells in 96-well plates and calculated using the Reed-Muench (1948) method. Toxicity controls were performed to show that the dilutions at which the compound prevented virus detection did not inhibit cell growth, which would also inhibit virus detection (no cells to replicate in). Virus replication controls without compound were included to show the level of inhibition by comparing treated samples to these controls. A concentration sampled four times for surviving virus and titered on appropriate cells for each test. Two independent trials were performed for each virus.

Results:

Toxicity. The “10%” compound was not cytotoxic in any virus test.

Rhinovirus 14 titers.

Results are summarized in Table 4. The preparation significantly reduced virus titers by a factor of 10,000 (4 logten drop) in trial 1 and by a factor of 100,000 in trial 2 down to below the limits of detection even when diluted to 3.2%. Even when diluted to 1%, there was 320-fold loss in virus titer (1.5-2 logten drop). The inhibition profile was dose responsive, which is usually a validation of a good assay.

Table 4. Titer of surviving Rhinovirus 14 after 15 minute liquid/liquid contact with test compounds (Log TCID50 per .18 mL).

SAMPLE ID Trial 1 Trial 2
MEM 4.0 5.0
ACS 200 10% <0.75a <0.75a
ACS 200 3.2% <0.75a <0.75a
ACS 200 1.0% 2.5 3.0
ACS 200 0.32% 4.0 4.3

Discussion:

It is likely that ACS 200 does not actually destroy RV as would bleach, because the enveloped viruses tested were not inactivated by the product as they would be with bleach, yet they are notoriously unstable and easy to disrupt. To grow in cell culture, RV is dependent on divalent cations such as MgCl2. These ions may enable the virus to interact with cells, may maintain capsid (protein structure surrounding virus RNA) stability, or the ions may act as cofactors that allow key virus enzymes responsible for virus replication to function [1]. Thus, it is feasible that the reason that rhinovirus was inhibited by ACS 200 was that the silver ion in the product interacted with the virus or its key enzymes to replace the Mg++ ion. Presumably, the silver ion inhibited by one of these three mechanisms. The only question is how permanent the interaction is, because compound to compound interactions (i.e., ion to protein) can be reversible. The ion binds and then detaches and may bind again and detach again. However, given the nature of the silver ion once free to bind, the silver ion binding to a virus protein or the capsid is likely an irreversible interaction with the virus.

Conclusions: Product ACS 200 profoundly inhibits rhinoviruses, but not any enveloped virus tested.

References:

1. Zhao R, Hadfield AT, Kremer MJ, Rossmann MG (1997) Cations in human rhinoviruses. Virology 227: 13-23.