Powassan Virucidal Assay Final Report

Antimicrobial Activity of Experimental Solutions Using Powassan (strain LB)
Test Solution: ACS 200
April 17, 2012

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

Department of Microbiology, Virology
Utah State University

Sponsor:                   Results RNA
Test start date:         17 Apr 2015
Test Compound:     ACS 200

Viruses:                                                                                 Cells:

Powassan (strain LB)                                                        Baby hamster kidney (BHK)

Cell Growth Medium:                Minimal Essential Medium (MEM) with 10% Fetal bovine serum

Virucidal Test medium:             MEM

Virus Propagation medium:

MEM + 2% FBS + 0.22% NaHCO3 + 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 “100%” compound was not cytotoxic in any virus test.

Powassan virus titers.  Results are summarized in Table 1.  The undiluted preparation (mixed 1:1 with virus) did significantly inactivate the virus.

Table 1.  Titer of surviving Powassan virus after 1 h liquid/liquid contact with test compound (Log TCID50 per .18 mL).

SAMPLE ID Trial 1
DMEM >9.5
ACS 200 100% 6.5
ACS 200 32% 8.5
ACS 200 10% 9.3
ACS 200 3.2% 8.3
ACS 200 1.0% 9.3
ACS 200 0.32% 8.5
ACS 200 0.1% 8.5
ACS 200 0.032% 9.3

 

Discussion:

It is likely that ACS 200 did inactivate the virus when used undiluted, but not at other dilutions.  Even though there was some reduction using ACS200 at 32%, 3.2%, 0.32%, and 0.1 %, these are not likely significantly different from the control virus titer.

Conclusions:  Undiluted ACS 200 reduced Powassan virus titers by 2 logten (100-fold) after a 1 h incubation at 37°C.  This suggests that ACS 200 can deactivate Powassan virus on contaminated surfaces.

Problems with Assay:

1- We did not reach an endpoint (where no virus was detectable) with the Powassan virus.  That means that the actual amount of virus could have been 10 logten or higher.  If so, some of the other dilutions could have significantly reduced virus titer (logten 8.5 now becomes a significant reduction).

2-The virus used had a very high titer, maybe too high to be biologically significant.

Solutions:

1-Redo assay to confirm results

2-Redo test using a lower amount of virus, i.e. 7 logten.