SC IEN

CE

United States

Environmental Protection

Agency

EPA 600/R-09/139 | November 2009 | www.epa.gov/ord

Freeze-Dried Vaccinia Virus

Persistence Testing and

Liquid Decontamination

Technology Evaluation

INVESTIGATION AND TECHNOLOGY 

EVALUATION REPORT

Office of Research and Development

National Homeland Security Research Center
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EPA/600/R-09/139 | November 2009 | www.epa.gov/ord

INVESTIGATION AND TECHNOLOGY 

EVALUATION REPORT

Freeze-Dried Vaccinia Virus Persistence 

Testing and Liquid Decontamination

Technology Evaluation

By

Young W. Choi, Morgan Q. Shaw, James V. Rogers, Daniel J. Chappie, Michael L. Taylor,

Karen B. Riggs, and Zachary J. Willenberg

Battelle

505 King Avenue

Columbus, Ohio 43201

Joseph P. Wood

Task Order Project Officer

National Homeland Security Research Center

Office of Research and Development

U.S. Environmental Protection Agency

Mail Code E343-06

Research Triangle Park, NC 27711

Office of Research and Development

National Homeland Security Research Center, Decontamination and Consequence Management Division
iv

Notice

The U.S. Environmental Protection Agency (EPA), through its Office of Research 

and Development’s National Homeland Security Research Center (NHSRC), funded, 

directed, and managed this investigation and technology evaluation through a 

Blanket Purchase Agreement under General Services Administration contract number 

GS23F0011L-3 with Battelle. This report has been peer and administratively reviewed 

and has been approved for publication as an EPA document. Mention of trade names or 

commercial products does not constitute endorsement or recommendation for use of a 

specific product.
v

Foreword

The U.S. Environmental Protection Agency (EPA) is charged 

by Congress with protecting the nation’s air, water, and land 

resources. Under a mandate of national environmental laws, 

EPA strives to formulate and implement actions leading to a 

compatible balance between human activities and the ability 

of natural systems to support and nurture life. To meet this 

mandate, the EPA’s Office of Research and Development 

(ORD) provides data and scientific support that can be used 

to solve environmental problems and to build the scientific 

knowledge base needed to manage our ecological resources 

wisely, to understand how pollutants affect our health, and to 

prevent or reduce environmental risks.

In September 2002, EPA announced the formation of the 

National Homeland Security Research Center (NHSRC). 

The NHSRC is part of the ORD; it manages, coordinates, 

and supports a variety of research and technical assistance 

efforts. These efforts are designed to provide appropriate, 

affordable, effective, and validated technologies and methods 

for addressing risks posed by chemical, biological, and 

radiological terrorist attacks. Research focuses on enhancing 

our ability to detect, contain, and clean up in the event of 

such attacks.

The NHSRC has developed the Technology Testing and 

Evaluation Program (TTEP) in an effort to provide reliable 

information regarding the performance of homeland 

security related technologies. TTEP provides independent, 

quality assured performance information that is useful 

to decision makers in purchasing or applying the tested 

technologies. It provides potential users with unbiased, 

third-party information that can supplement vendor-provided 

information. Stakeholder involvement ensures that user 

needs and perspectives are incorporated into the test design 

so that useful performance information is produced for each 

of the tested technologies. The technology categories of 

interest include detection and monitoring, water treatment, air 

purification, decontamination, and computer modeling tools 

for use by those responsible for protecting buildings, drinking 

water supplies and infrastructure.

The evaluation reported herein was conducted by Battelle 

as part of TTEP. Information on NHSRC and TTEP can be 

found at http://www.epa.gov/nhsrc/ttep.html.
vi

Acknowledgments

The authors wish to acknowledge the support of all those who helped plan and conduct 

the evaluation, analyze the data, and prepare this report. We also would like to thank 

Dr. Timothy Dean of the EPA National Risk Management Research Laboratory - 

Air Pollution Prevention and Control Division, Dr. Sarah Taft of the EPA NHSRC, 

and Dr. Michael Ottlinger of the EPA Office of Emergency Management – National 

Decontamination Team for their reviews of this report.
vii

Contents

Notice.............................................................................................................................................................................iv

Foreword.........................................................................................................................................................................v

Acknowledgments..........................................................................................................................................................vi

Abbreviations/Acronyms...............................................................................................................................................ix

Executive Summary........................................................................................................................................................x

1.0 Introduction............................................................................................................................................................1

2.0 Persistence Testing.................................................................................................................................................3

2.1 Test Materials...............................................................................................................................................3

2.2 Vaccinia Virus Recovery from Test Materials..............................................................................................3

2.2.1 Spiking the Coupons and Freeze-Drying ...........................................................................................3

2.2.2 Vaccinia Virus Extraction and Quantification.....................................................................................3

2.2.3 Vaccinia Virus Recovery Results........................................................................................................3

2.3 Persistence Testing Approach.......................................................................................................................4

2.4 Test Results...................................................................................................................................................6

2.4.1 Glass...................................................................................................................................................6

2.4.2 Galvanized Metal................................................................................................................................9

2.4.3 Painted Cinder Block........................................................................................................................11

2.4.4 Industrial Carpet ...............................................................................................................................14

2.4.5 Comparison of Persistence Testing Results......................................................................................16

3.0 Decontamination Technology Evaluation............................................................................................................17

3.1 Technology Descriptions............................................................................................................................17

3.2 Test Matrix..................................................................................................................................................17

3.3 Technology Evaluation...............................................................................................................................18

4.0 Quality Assurance/Quality Control......................................................................................................................21

4.1 Equipment Calibration ...............................................................................................................................21

4.2 Audits .........................................................................................................................................................21

4.2.1 Performance Evaluation Audit .........................................................................................................21

4.2.2 Technical Systems Audit .................................................................................................................21

4.2.3 Data Quality Audit............................................................................................................................21

4.3 QA/QC Reporting .....................................................................................................................................21

4.4 Data Review ...............................................................................................................................................21

4.5 Deviations...................................................................................................................................................21

5.0 Summary..............................................................................................................................................................23

5.1 Vaccinia Virus Persistence..........................................................................................................................23

5.2 Vaccinia Virus Liquid Decontamination ...................................................................................................23

6.0 References............................................................................................................................................................25
viii

Tables

Table ES-1. Summary of Persistence Test Conditions and Vaccinia Virus Recoveries............................................xi

Table 2-1. Test Materials.........................................................................................................................................3

Table 2-2. Vaccinia Virus Recoveries.....................................................................................................................4

Table 2-3. Persistence Tests Matrix ........................................................................................................................5

Table 2-4. Vaccinia Virus Persistence on Glass......................................................................................................7

Table 2-5. Vaccinia Virus Persistence on Galvanized Metal ..................................................................................9

Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block...........................................................................12

Table 2-7. Vaccinia Virus Persistence on Industrial Carpet..................................................................................14

Table 2-8. Summary of Vaccinia Virus Persistence ..............................................................................................16

Table 3-1. Decontamination Technology Evaluation Matrix................................................................................17

Table 3-2. Decontamination Efficacy against Vaccinia Virus on Galvanized Metal ............................................18

Table 3-3. Decontamination Efficacy against Vaccinia Virus on Industrial Carpet..............................................19

Table 3-4. Summary of Decontamination Efficacy against Vaccinia Virus..........................................................19

Table 4-1. Performance Evaluation Audit Results................................................................................................21
ix

Abbreviations/Acronyms

AOAC Association of Official Analytical Chemists

ASTM American Society for Testing and Materials

ATCC American Type Culture Collection

°C degrees Celsius

CaCO3 calcium carbonate

cm centimeter

CO2 carbon dioxide

D/E broth Dey and Engley broth

EPA U.S. Environmental Protection Agency

g gram

MDCK Madin-Darby canine kidney

MEM minimum essential medium

mL milliliter

NHSRC National Homeland Security Research Center 

ORD EPA Office of Research and Development

PBS phosphate-buffered saline

PFU plaque-forming unit

ppm parts per million

QA quality assurance

QC quality control

QMP quality management plan

RH relative humidity

rpm revolutions per minute

TSA technical systems audit

TTEP Technology Testing and Evaluation Program 

μL microliter
x

Executive Summary

The U.S. Environmental Protection Agency’s (EPA) National 

Homeland Security Research Center (NHSRC) Technology 

Testing and Evaluation Program (TTEP) helps to protect 

human health and the environment from adverse impacts of 

terrorist acts by carrying out performance tests on homeland 

security technologies. Under TTEP, the persistence of 

viable vaccinia virus (surrogate for the variola virus, which 

causes smallpox) that was freeze-dried on coupons prepared 

from glass, galvanized metal, painted cinder block, and 

industrial carpet was investigated. The efficacy of two 

liquid decontamination technologies for inactivating the 

vaccinia virus that was freeze-dried on coupons of galvanized 

metal and industrial carpet was evaluated at room and low 

temperatures.

For persistence testing, materials were “contaminated” by 

spiking with a target level of 1 x 107 plaque-forming units 

(PFUs) of the vaccinia virus per coupon and immediately 

freeze-dried. The persistence of the vaccinia virus was 

subsequently investigated for up to four test durations at 

four environmental conditions comprised of variations 

in temperature and relative humidity (RH). The four 

environmental conditions (the four combinations of low/high 

RH, and low and ambient temperature) were selected for 

testing to investigate the effect of RH and temperature

on persistence, and are as follows:

■ Room temperature, low RH

○ Test durations: 14, 21, 28, and 42 days 

■ Room temperature, high RH

○ Test durations: 1, 3, 7, and 14 days

■ Low temperature, low RH

○ Test durations: 14, 21, 28, and 56 days

■ Low temperature, high RH

○ Test durations: 7, 14, 21, and 42 days

Under the “room temperature, high RH” environmental 

condition, the vaccinia virus remained viable for three days 

on glass and one day on galvanized metal and painted cinder 

block; the vaccinia virus was not detected on industrial 

carpet after one day. Under the “low temperature, high RH” 

environmental condition, the vaccinia virus remained viable 

for at least seven days on all materials.

The vaccinia virus generally remained viable longer under 

low RH conditions at both room and low temperatures 

compared to the respective tests conducted at a high 

RH. The vaccinia virus persisted on all four materials 

for at least 14 days at the “room temperature, low RH” 

environmental condition; the vaccinia virus persisted 

for 42 days on glass, galvanized metal, and painted 

cinder block. Under the “low temperature, low RH” 

environmental condition, the vaccinia virus persisted on 

all four materials for the 56-day duration. A summary 

of the actual test conditions and PFU values recovered 

from each of the materials is provided in Table ES-1.

The liquid decontamination technologies evaluated were 

1% citric acid and hospital grade quaternary ammonium 

salt (732 ppm) disinfectant; these common chemicals were 

chosen because they are widely available for use. The 

evaluation was conducted at room and low temperatures with 

a 30-minute liquid decontaminant contact time. Both liquid 

decontamination technologies were applied to galvanized 

metal and industrial carpet coupons inoculated with the 

vaccinia virus and freeze-dried.

Neither 1% citric acid nor 732 ppm quaternary ammonium 

salt reduced the vaccinia virus to non-detectable levels. For 

1% citric acid, mean log reductions in vaccinia virus PFUs 

on galvanized metal were 3.2 at both the room and low 

temperatures; on industrial carpet, log reductions were 2.6 at 

room temperature and 2.5 at low temperature. When 732 ppm 

quaternary ammonium salt was used, mean log reductions in 

vaccinia virus PFUs on galvanized metal were 1.5 at both the 

room and low temperature conditions; on industrial carpet the 

log reductions were less than 1.0.
xi

Table ES-1. Summary of Persistence Test Conditions and Vaccinia Virus Recoveriesa

Environmental Condition / 

Test Duration

Temperature 

(°C)b RH (%)b

Mean Recovered Vaccinia Virus (PFUs)

Glass

Galvanized 

Metal

Painted 

Cinder Block

Industrial 

Carpet

Room Temperature, Low RH

14 Days 22 10 2.43 x 107 1.90 x 107 2.57 x 107 4.34 x 106

21 Days 23 1 2.33 x 104 1.49 x 104 5.46 x 100 ND

28 Days 23 1 2.45 x 101 1.11 x 104 5.81 x 103 ND

42 Days 23 1 4.22 x 103 5.30 x 103 4.00 x 10-1 c ND

Room Temperature, High RH

1 Day 21 89 3.40 x 100 c 1.66 x 101 2.55 x 104 ND

3 Days 23 93 6.93 x 103 ND ND Not Tested

7 Days 23 98 ND ND ND ND

14 Days 21 89 ND ND ND ND

Low Temperature, Low RH

14 Days 7 7 6.04 x 107 2.71 x 107 6.27 x 107 1.24 x 107

21 Days 8d 8d 3.45 x 106 1.90 x 106 3.84 x 106 9.92 x 105

28 Days 7e 7e 4.05 x 106 3.04 x 106 3.06 x 106 1.07 x 106

56 Days 7 1 2.87 x 104 5.38 x 104 2.99 x 104 1.55 x 101

Low Temperature, High RH

7 Days 7 90 5.22 x 106 1.47 x 103 c 7.68 x 106 1.01 x 106

14 Days 7 101 3.26 x 103 c ND 2.54 x 105 1.32 x 105

21 Days 6e 95e ND ND 1.49 x 105 3.72 x 104

42 Days 7 96 ND ND ND ND

a Vaccinia virus was applied to the coupons as an aqueous suspension, the coupons were freeze-dried, then placed in the environmental condition 

for the test duration. Spike amounts ranged from 6.98 x 106 to 4.79 x 107 PFUs. b

 Temperature and RH values are the means based on continuous monitoring at 1-to-6-minute intervals.

c Vaccinia virus was not detected on some of the replicate test coupons; a value of 0 PFUs was used for non-detects in calculation of mean 

vaccinia virus recovery.

d Temperature and RH were recorded for less than one day after test initiation due to unplanned shut-down/failure of data-loggers. No unusual 

events occurred that would lead one to expect that the temperature and RH deviated from those of comparable tests. 

e Temperature and RH were recorded for approximately 15 days after test initiation. No unusual events occurred that would lead one to expect that 

the temperature and RH deviated from those of comparable tests.

ND = Not detected; the detection limit is 10 PFUs.
xii

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1

1.0

Introduction

NHSRC’s TTEP works in partnership with recognized testing 

organizations; with stakeholder groups consisting of buyers, 

vendor organizations, scientists, and permitters; and with 

participation of individual technology developers in carrying 

out performance tests on homeland security technologies. 

In response to the needs of stakeholders, TTEP investigates 

the natural persistence of biological and chemical agents 

and evaluates the performance of innovative homeland 

security technologies by developing test plans, conducting 

evaluations, collecting and analyzing data, and preparing 

peer-reviewed reports. All evaluations are conducted in 

accordance with rigorous quality assurance (QA) protocols 

to ensure the generation of high quality data and defensible 

results. TTEP provides unbiased, third-party information 

supplementary to vendor-provided information that is useful 

to decision makers in purchasing or applying the evaluated 

technologies. Stakeholder involvement ensures that user 

needs and perspectives are incorporated into the evaluation 

design to produce useful performance information for each 

evaluated technology. 

Under TTEP, persistence of viable vaccinia virus was tested 

after applying the vaccinia virus as an aqueous suspension 

to the surface of a test material which was then freeze-dried. 

The performance of liquid technologies for decontaminating 

freeze-dried vaccinia virus on various materials was also 

evaluated. The vaccinia virus is used in the smallpox 

vaccine and is a commonly used surrogate for variola virus, 

the etiological agent of smallpox. Variola virus is easily 

transportable in a temperature-stable freeze-dried state, and 

it is conceivable that variola virus might be aerosolized 

to cause infection(1). The persistence of the vaccinia virus 

was investigated under various environmental conditions, 

after initially being freeze-dried on glass, galvanized metal, 

painted cinder block, and industrial carpet. The ability of 

two liquid decontamination technologies (1% citric acid 

and 732 ppm quaternary ammonium salt) to inactivate the 

vaccinia virus after the virus was initially freeze-dried on 

galvanized metal and industrial carpet, and subsequently 

held under room and low temperature conditions was also 

evaluated.

The persistence investigation and liquid decontamination 

technology evaluations were conducted according to a peerreviewed test/QA plan(2) that was developed according to 

the requirements of the TTEP quality management plan 

(QMP)(3). The persistence tests and decontamination 

evaluations used the PFU assay with Vero (African Green 

Monkey kidney) cells to quantify the viable vaccinia virus 

extracted from test materials. This report documents the log 

reductions in PFUs associated with a natural reduction in the 

viable vaccinia virus under various environmental conditions 

and the decrease in the viable vaccinia virus exposed to two 

liquid decontamination technologies. 
2

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3

2.0

Persistence Testing

2.1 Test Materials

Materials used for the vaccinia virus persistence testing 

are described in Table 2-1. Test coupons were uniformly 

cut to 1.9 cm width x 7.5 cm length from larger pieces of 

material. Edges and damaged areas were avoided in cutting 

test coupons. The coupons were then autoclaved or gammairradiated (Table 2-1) and stored under sterile conditions 

until use, which was intended to minimize microorganism 

contamination of the coupons’ surfaces. All of the materials 

used for the vaccinia virus persistence testing have been used 

in previous TTEP testing.

2.2 Vaccinia Virus Recovery from Test Materials

2.2.1 Spiking the Coupons and Freeze-Drying

Each coupon was spiked with a target level of 1 x 107 PFUs 

of the vaccinia virus [American Type Culture Collection 

(ATCC) VR119] per coupon (i.e., 100 μL of 1 x 108 PFUs/

mL stock suspension per coupon). Spiking of the coupons 

was performed using a multichannel micropipette as two 

rows of five droplets (10 μL per droplet) across the surface of 

the test coupon.

After spiking, the coupons were frozen overnight at ≤-80°C 

in preparation for the freeze-drying process. Using a VirTis 

DBT Benchtop 3.5 L Freeze-Dryer, the spiked, frozen 

coupons were freeze-dried until the samples were visibly dry 

(approximately two to four hours).

2.2.2 Vaccinia Virus Extraction and Quantification

At the completion of the freeze-drying process (time zero), 

the coupons were extracted and assayed for PFUs. The 

extraction approach involved placing each individual coupon 

in a 50 mL conical vial to which 10 mL of sterile extraction 

buffer [i.e., phosphate-buffered saline (PBS)] was added. 

The tubes were agitated on an orbital shaker for 15 minutes 

at approximately 200 revolutions per minute (rpm) at room 

temperature. Each coupon extract was then serially diluted 

in PBS (10-fold dilutions through 10-7). An aliquot (0.1 mL) 

of the undiluted extract and each serial dilution was plated 

onto monolayers of Vero cells. The tissue culture plates 

were rocked every 15 minutes for approximately one hour 

at ~37°C, to allow adsorption of the vaccinia virus to the 

Vero cells. The cultures were then overlaid with minimum 

essential medium (MEM) containing 2% fetal bovine serum, 

0.5% methylcellulose, and antibiotics. The cultures were 

incubated for 72 ± 4 hours at 37 ± 2°C in 95% air and 5% 

CO2. Following incubation, crystal violet dye was added to 

the monolayers for 15 minutes, removed and the cells were 

rinsed with PBS. Plaques were visualized as clearings in the 

purple monolayer of Vero cells. The plaques were counted 

manually and the number of PFUs/mL was determined by 

multiplying the mean number of plaques per well by the 

reciprocal of the dilution. The plaque assay has a detection 

limit of 10 PFUs.

2.2.3 Vaccinia Virus Recovery Results

Prior to persistence testing, efforts were conducted to 

determine if an acceptable level of the vaccinia virus (>1% 

of the applied inoculum per the test/QA plan(2)) could be 

recovered from the coupons. The coupons were spiked and 

freeze-dried as described in the preceding sections. Then the 

vaccinia virus was extracted from the coupons and analyzed 

for PFUs. Initial determinations of vaccinia virus recoveries 

were conducted with three replicate coupons per material. 

As acceptable recoveries were obtained, final demonstrations 

using five replicate coupons were used to obtain mean 

Table 2-1. Test Materials

Material

Lot, Batch, or ASTM No., or 

Observation

Manufacturer/ 

Supplier Name

Approximate Coupon 

Size, width x length

Approximate 

Coupon 

Thickness

Material 

Preparation

Glass C1036 Brooks Brothers 1.9 cm x 7.5 cm 0.3 cm Autoclaved

Galvanized Metal Industry heating, ventilation, 

and air conditioning standard 

24 gauge galvanized steel

Accurate 

Fabrication

1.9 cm x 7.5 cm 0.06 cm Cleaned with 

acetone; 

autoclaved

Painted Cinder 

Block (concrete)

ASTM C90. Brush and roller 

painted all sides. One coat 

Martin Senour latex primer 

(#71-1185) and one coat 

Porter Paints latex semi-gloss 

finish (#919)

Wellnitz 1.9 cm x 7.5 cm 0.5 cm Autoclaved

Industrial Carpet ShawTek, EcoTek 6 Color: 

mottled gray/dark brown/ black

Shaw 

Industries, Inc.

1.9 cm x 7.5 cm 0.7 cm Gamma 

Irradiation
4

recovery and variance data. The vaccinia virus recovery 

results are provided in Table 2-2. Acceptable vaccinia virus 

recoveries were obtained for all materials used in persistence 

testing (glass, galvanized metal, painted cinder block, and 

industrial carpet). The percent vaccinia virus recoveries were 

higher when three replicates were used as compared to five 

replicates for glass, galvanized metal, and industrial carpet. 

Testing was conducted on two different days, and the higher 

inoculum associated with the five replicates contributed 

to lower percent recoveries since comparable amounts of 

vaccinia virus (PFUs) were recovered from the three and five 

replicates per material.

Mean percent vaccinia virus recoveries were calculated based 

on the mean of the replicate vaccinia virus percent recoveries 

per material and test (tests with three replicates and five 

replicates were conducted on different days) as:

Equation 2-1.

n

Q

P

jk

n

i

ijk

P

∑=





 





×















= 1

100 %

Where:

Pjk = mean percent vaccinia virus recovery for the j

th

material and kth test

Pijk = vaccinia virus recovered for the i

th replicate, jth

material and kth test (in PFUs)

Q = inoculum amount (in PFUs)

∑=





 





×













 n

i

ijk

Q

P

1

100% = sum of the replicate percent 

vaccinia virus recoveries based on 

the amount of vaccinia virus 

recovered (Pijk) and the inoculum 

amount (Q)

n = number of replicate test coupons (three or five).

If no PFUs were detected for a test coupon, a value of 0 was 

substituted for the vaccinia virus recovery (P).

2.3 Persistence Testing Approach

The vaccinia virus was quantified from the liquid extracts 

obtained from the test coupons (spiked coupons placed in 

the exposure chamber with temperature, RH, and contact 

time treatments) and positive controls (spiked coupons, 

freeze-dried, and extracted at time zero [at the completion 

of freeze-drying]). Spiking and freeze-drying of the coupons 

and vaccinia virus extraction and quantification followed 

the approach described in Section 2.2.1. The log reduction 

in the vaccinia virus viability (quantified by determining 

PFUs) was calculated as N/N' where N is the mean number of 

PFUs from the five positive controls of a given material and 

environmental condition and N′ is the number of PFUs from 

each test coupon replicate of a given material, environmental 

condition, and contact time. The log reduction in PFUs for 

each individual test coupon (R) was calculated for each of the 

five replicate test coupons of each material, environmental 

condition, and contact time as:

Equation 2-2.





















= N ijkl

N R ijkl

jk

' log10

Where:

Rijkl = log reduction in PFUs for the i

th replicate test coupon, 

j

th material, kth environmental condition, 

and l

th contact time

Njk =arithmetic mean PFU from the five positive controls 

for the j

th material and kth environmental condition

N'ijkl = PFUs recovered on the i

th replicate test coupon, 

j

th material, kth environmental condition, and lth

contact time.

Table 2-2. Vaccinia Virus Recoveries

Material / Replicates Inoculum (PFUs) PFU Detectionsa Recovered Virus (PFUs)b % Virus Recoveryb

Glass

3 replicates

5 replicates

5.95 x 107

1.67 x 108

3/3

5/5

3.05 ± 0.73 x 107

2.37 ± 0.16 x 107

85 ± 12

24 ± 0.97

Galvanized Metal

3 replicates

5 replicates

5.95 x 107

1.67 x 108

3/3

5/5

0.54 ± 2.39 x 107

1.90 ± 0.66 x 107

90 ± 40

57 ± 4.0

Painted Cinder Block

3 replicates

5 replicates

5.95 x 107

1.67 x 108

3/3

5/5

6.26 ± 4.09 x 106

4.05 ± 0.48 x 106

18 ± 6.9

24 ± 2.9

Industrial Carpet

3 replicates

5 replicates

5.95 x 107

1.67 x 108

3/3

5/5

1.52 ± 0.13 x 107

7.17 ± 0.84 x 106

43 ± 2.2

4.3 ± 0.50

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons. 

b

 Data are expressed as mean ± standard deviation.
5

If no PFUs were detected for a test coupon (N'), the value 

“1” was substituted for N'. Since the value 1 is greater than 

the observed value of 0, the estimate with this substitution 

becomes a lower bound for the true log reduction. Next, the 

mean log reduction in PFUs (R) for the five replicate test 

coupons of a given material and environmental condition is 

calculated as:

Equation 2-3.

n

jkl

n

i

Rijkl R

∑= = 1

Where:

Rjkl = mean log reduction in PFUs for the j

th material, 

kth environmental condition, and l

th contact time

∑=

n

i

Rijkl 1

= sum of the log reductions in PFUs for each 

individual test coupon (i) for the j

th material,

kth environmental condition, and l

th contact time

n = number of test coupon replicates (five).

The test matrix and various test conditions that were utilized 

for the vaccinia virus persistence testing are summarized in 

Table 2-3. The environmental conditions included various 

combinations of temperature and RH. Persistence was 

measured for four types of test coupons: glass, galvanized 

metal, painted cinder block, and industrial carpet, and the test 

durations ranged from 1 to 56 days. Initial time points were 

selected based on comparable data available in the literature 

(i.e., Stone et al., 2006(4)), but subsequent time points were 

adaptively chosen (i.e., shorter or longer durations) based 

on the initial test results, and tests were not conducted 

sequentially from the shortest to longest test duration. Please 

note that when possible multiple tests were initiated on the 

same day such that only one set of positive controls would be 

used per material (i.e., the same inoculum stock was used for 

all tests initiated on the same day). 

Similar vaccinia virus (although not freeze-dried) 

persistence testing was previously conducted under TTEP, 

using galvanized metal and painted cinder block under 

environmental conditions of ~20°C and 40%-70% RH, and 

at 30°C at either high (>70%) or low RH (<40%) for up to 14 

days(4). In that study, the vaccinia virus persisted longer when 

exposed to low RH. The current testing was conducted at two 

temperatures (room and low) and two RH levels (high and 

low) to evaluate the influence of temperature and RH on the 

persistence of the vaccinia virus following the freeze-drying 

Table 2-3. Persistence Tests Matrixa

Target Environmental Condition / Test Duration

Temperature (°C) RH (%)

Mean Range Mean Range

Room Temperature (22°C), Low RH (20%)

14 Days 21.9 20.44 - 25.48 10.1 9.51 - 44.34

21 Days 22.6 21.58 - 23.47 1.0 1.00 - 39.25

28 Days 23.0 22.15 - 24.41 1.0 1.00 - 40.33

42 Days 23.1 22.42 - 24.12 1.0 1.00 - 31.57

Room Temperature (22°C), High RH (70%)

1 Day 21.3 20.94 - 23.54 89.2 35.64 - 92.74

3 Daysa 23.0 22.54 - 25.28 93.0 21.53 - 97.51

7 Days 22.8 21.32 - 23.33 97.9 41.50 - 100.88

14 Days 21.3 19.46 - 25.14 89.1 39.29 - 92.80

Low Temperature (10°C), Low RH (20%)

14 Days 7.1 6.36 - 24.15 7.2 6.37 - 39.77

21 Days 7.6b 5.90b - 25.60b 7.7b 5.68b - 39.06b

28 Days 7.0c 6.61c - 24.87c 7.4c 6.46c - 40.51c

56 Days 7.0 6.64 - 24.73 1.0 1.00 - 39.02

Low Temperature (10°C), High RH (70%)

7 Days 6.9 5.77 - 23.81 90.4 43.91 - 93.50

14 Days 7.5 7.14 - 23.81 100.6 90.39 - 102.20

21 Days 6.1c 5.80c - 25.89c 94.8c 37.11c - 97.34c

42 Days 6.6 6.31 - 23.50 95.8 46.21 - 97.38

a Test materials included glass, galvanized metal, painted cinder block, and industrial carpet, with the exception that industrial carpet was not 

tested during the 3-day duration at room temperature, high RH.

b

 Temperature and RH were recorded for less than one day after test initiation. 

c

 Temperature and RH were recorded for approximately 15 days after test initiation.
6

process. The actual temperatures and RH levels associated 

with each test are provided in Table 2-3.

Test coupons were placed inside sealed Lock&LockTM plastic 

storage containers. For low RH conditions, Drierite (W.A. 

Hammond Drierite Co.) was added to the containers, and 

for high RH conditions, 70% RH humidity beads (Heartfelt 

Industries) were added to the containers. For testing at 

the low temperature, the containers were placed inside 

a refrigerator. No special manipulations for controlling 

temperature were required for the room temperature testing. 

Temperature and RH were measured and the data recorded 

continuously at one-to-six-minute intervals with a HOBO 

U10 data logger (Onset Computer Corporation), except as 

noted on Table 2-3. Starting from ambient RH levels between 

approximately 35-45%, the RH tended to rapidly decrease 

during low RH tests and steadily increase during high RH 

tests, due to the Drierite and humidity beads, respectively. 

The range of RH values monitored included these relatively 

short acclimation periods during which the RH level (starting 

at ambient level) was decreasing or increasing within the 

storage containers. As such, occasionally the range of RH 

levels listed in Table 2-3 overlapped depending on the 

starting ambient level.

2.4 Test Results

Persistence results for each material/environmental condition 

combination are summarized in Tables 2-4 through 2-7. 

Several tests (e.g., the 21-, 28-, and 42-day tests at the “room 

temperature, low RH” environmental condition for glass 

on Table 2-4) were initiated on the same day so the same 

positive controls were applicable for each test. The amount 

of PFUs in the inoculums (as quantified by spike controls and 

presented as the inoculums for the positive and test coupons) 

ranged from 6.98 x 106 to 4.79 x 107 PFUs per coupon due 

to the varying amounts of the vaccinia virus titer propagated, 

and all calculations were based on the recorded results. A 

summary of vaccinia virus persistence results is provided in 

Table 2-8.

2.4.1 Glass

The vaccinia virus persistence results on glass are 

summarized in Table 2-4. With the exception of the “room 

temperature, high RH” environmental condition, the 

vaccinia virus remained viable for ≥14 days on glass. The 

vaccinia virus was found to remain viable on glass (with 

≤3.9 mean log reductions in PFUs) for 42 days under the 

“room temperature, low RH” environmental condition, 56 

days under the “low temperature, low RH” environmental 

condition, and seven days under the “low temperature, high 

RH” environmental condition. 

The vaccinia virus was least viable on glass with high RH 

conditions at both room and low temperatures. For the “low 

temperature, high RH” environmental condition, a 0.9 mean 

log reduction in PFUs occurred following the 7-day duration, 

while durations of 14, 21, and 42 days resulted in ≥5.6 

mean log reductions in PFUs. At the “room temperature, 

high RH” environmental condition, the vaccinia virus was 

viable after one and three days but with ≥4.3 mean log 

reductions in PFUs; the vaccinia virus was not detected at 

the 7- and 14-day durations. At the “low temperature, low 

RH” environmental condition, the virus recovery at 14 days 

was 126% (no outliers were identified as being outside three 

standard deviations of the mean). Recovery greater than 

100% may be indicative of day-to-day variability within the 

extraction procedure and assay. Test coupons showing greater 

than 100% recovery or having a negative log reduction, 

especially at the low temperature, low RH environmental 

condition, are an indication of minimal inactivation of the 

vaccinia virus and not necessarily viral propagation or an 

error in measurement. 
7

Table 2-4. Vaccinia Virus Persistence on Glass

Test Duration / 

Sample Inoculum (PFUs) PFU Detectionsa Recovered Virus (PFUs)b

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH

14 Days

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

3.72 x 107

3.72 x 107

0

0

5/5

5/5

0/1

0/1

3.75 ± 0.15 x 107

2.43 ± 0.64 x 107

ND

ND

101 ± 4.02

65.4 ± 17.1

-

-

-

0.2 ± 0.1

-

-

21 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

2.33 ± 2.34 x 104

ND

ND

36.3 ± 21.4

0.33 ± 0.33

-

-

-

2.6 ± 1.2

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

2.45 ± 0.74 x 101

ND

ND

36.3 ± 21.4

0.00 ± 0.00

-

-

-

5.0 ± 0.16

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

4.22 ± 5.83 x 103

ND

ND

36.3 ± 21.4

0.07 ± 0.09

-

-

-

3.9 ± 1.4

-

-

Room Temperature, High RH

1 Day

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

3/5

0/1

0/1

2.53 ± 1.49 x 106

3.40 ± 4.82 x 100 g

ND

ND

36.3 ± 21.4

0.00 ± 0.00g

_

_

-

6.1 ± 0.5g

-

-

3 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

6.93 ± 10.1 x 103

ND

ND

36.3 ± 21.4

0.10 ± 0.14

-

-

-

4.3 ± 2.1

-

-

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.53 ± 1.49 x 106

NDg

ND

ND

36.3 ± 21.4

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

3.72 x 107

3.72 x 107

0

0

5/5

0/5

0/1

0/1

3.75 ± 0.15 x 107

NDg

ND

ND

101 ± 4.02

0.00 ± 0.00g

-

-

-

7.6 ± 0.0g

-

-
8

Table 2-4. Vaccinia Virus Persistence on Glass (continued)

Test Duration / 

Sample Inoculum (PFUs) PFU Detectionsa Recovered Virus (PFUs)b

% Virus 

Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

3.75 ± 0.15 x 107

6.04 ± 4.03 x 107

ND

ND

78.2 ± 3.12

126 ± 84.2

-

-

-

-0.2 ± 0.2

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

3.45 ± 1.09 x 106

ND

ND

36.3 ± 21.4

49.5 ± 15.6

-

-

-

-0.1 ± 0.1

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

4.05 ± 0.85 x 106

ND

ND

36.3 ± 21.4

58.1 ± 12.1

-

-

-

-0.2 ± 0.1

-

-

56 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.53 ± 1.49 x 106

2.87 ± 2.21 x 104

ND

ND

36.3 ± 21.4

0.41 ± 0.32

-

-

-

2.0 ± 0.3

-

-

Low Temperature, High RH

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

3.75 ± 0.15 x 107

5.22 ± 1.96 x 106

ND

ND

78.2 ± 3.12

10.9 ± 4.09

-

-

-

0.9 ± 0.2

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

1/5

0/1

0/1

2.53 ± 1.49 x 106

3.26 ± 7.29 x 103 g

ND

ND

36.3 ± 21.4

0.05 ± 0.10g

-

-

-

5.6 ± 1.9g

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.53 ± 1.49 x 106

ND g

ND

ND

36.3 ± 21.4

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.53 ± 1.49 x 106

NDg

ND

ND

36.3 ± 21.4

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls are inoculated, frozen overnight at ≤-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the 

completion of freeze-drying).

d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.

e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).

f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.

g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for 

non-detects in the calculation of mean log reduction.

h Temperature and RH were actually unknown.

ND = Not detected; the plaque assay detection limit is 10 PFUs.

“-” Not applicable.
9

2.4.2 Galvanized Metal

The vaccinia virus persistence results on galvanized metal 

are summarized in Table 2-5. Under both low RH conditions 

(at room and low temperature), the vaccinia virus remained 

viable for ≥42 days with the highest mean log reduction 

in PFUs of 3.9 occurring after the 42-day duration at the 

“room temperature, low RH” environmental condition. After 

56 days in the “low temperature, low RH” environmental 

condition, there was a 1.7 mean log reduction in PFUs.

The vaccinia virus did not remain as viable on galvanized 

metal under high RH conditions (at room or low temperature) 

compared to the low RH conditions, with ≥5.3 mean log 

reductions over all test durations. The vaccinia virus was 

only detected after one day in the “room temperature, 

high RH” environmental condition and after 7 days in the 

“low temperature, high RH” environmental condition on 

galvanized metal.

Table 2-5. Vaccinia Virus Persistence on Galvanized Metal

Test Duration / 

Sample

Inoculum 

(PFUs) PFU Detectionsa

Recovered Virus 

(PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH

14 Days

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

3.72 x 107

3.72 x 107

0

0

5/5

5/5

0/1

0/1

1.99 ± 0.16 x 107

1.90 ± 0.49 x 107

ND

ND

53.4 ± 4.36

51.0 ± 13.2

-

-

-

0.0 ± 0.1

-

-

21 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

1.49 ± 1.86 x 104

ND

ND

33.6 ± 15.6

0.21 ± 0.27

-

-

-

3.4 ± 1.9

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

1.11 ± 0.64 x 104

ND

ND

33.6 ± 15.6

0.16 ± 0.09

-

-

-

2.7 ± 1.1

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

5.30 ± 7.22 x 103

ND

ND

33.6 ± 15.6

0.08 ± 0.10

-

-

-

3.9 ± 1.5

-

-

Room Temperature, High RH

1 Day

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

1.66 ± 1.04 x 101

ND

ND

33.6 ± 15.6

0.00 ± 0.00

-

-

-

5.3 ± 0.4

-

-

3 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.34 ± 1.09 x 106

NDg

ND

ND

33.6 ± 15.6

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.34 ± 1.09 x 106

NDg

ND

ND

33.6 ± 15.6

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

3.72 x 107

3.72 x 107

0

0

5/5

0/5

0/1

0/1

1.99 ± 0.16 x 107

NDg

ND

ND

53.4 ± 4.36

0.00 ± 0.00g

-

-

-

7.3 ± 0.0g

-

-
10

 Table 2-5. Vaccinia Virus Persistence on Galvanized Metal (continued)

Test Duration / 

Sample Inoculum (PFUs) PFU Detectionsa

Recovered Virus 

(PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

1.99 ± 0.16 x 107

2.71 ± 0.42 x 107

ND

ND

41.5 ± 3.39

56.6 ± 8.77

-

-

-

-0.1 ± 0.1

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

1.90 ± 0.42 x 106

ND

ND

33.6 ± 15.6

27.2 ± 6.02

-

-

-

0.1 ± 0.1

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

3.04 ± 0.77 x 106

ND

ND

33.6 ± 15.6

43.6 ± 11.0

-

-

-

-0.1 ± 0.1

-

-

56 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

2.34 ± 1.09 x 106

5.38 ± 2.64 x 104

ND

ND

33.6 ± 15.6

0.77 ± 0.38

-

-

-

1.7 ± 0.3

-

-

Low Temperature, High RH

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

4/5

0/1

0/1

1.99 ± 0.16 x 107

1.47 ± 3.28 x 103 g

ND

ND

41.5 ± 3.39

0.00 ± 0.01g

-

-

-

6.6 ± 1.8g

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.34 ± 1.09 x 106

NDg

ND

ND

33.6 ± 15.6

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.34 ± 1.09 x 106

NDg

ND

ND

33.6 ± 15.6

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

2.34 ± 1.09 x 106

NDg

ND

ND

33.6 ± 15.6

0.00 ± 0.00g

-

-

-

6.4 ± 0.0g

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons. 

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls are inoculated, frozen overnight at ≤-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the 

completion of freeze-drying).

d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.

e Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).

f Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.

g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for 

non-detects in the calculation of mean log reduction.

h Temperature and RH were actually unknown.

ND = Not detected; the plaque assay detection limit is 10 PFUs.

“-” Not applicable.
11

2.4.3 Painted Cinder Block

The vaccinia virus persistence results on painted cinder block 

are summarized in Table 2-6. The vaccinia virus persisted 

for at least 42 days under low RH conditions (at both room 

and low temperatures). At the “low temperature, low RH” 

environmental condition, the mean log reductions in PFUs 

on painted cinder block were ≤2.2 for all test durations, 

including the 56-day duration. The vaccinia virus was also 

detected from all durations tested at the “room temperature, 

low RH” environmental condition, but log reductions were 

≥4.2 at 21, 28, and 42 days.

The vaccinia virus did not persist as long on painted 

cinder block under high RH conditions (room and low 

temperatures), compared to the respective low RH conditions. 

The vaccinia virus was detected after the 21-day duration, 

but not the 42-day duration, under the “low temperature, 

high RH” environmental condition. The vaccinia virus was 

detected only at one day for the “room temperature, high 

RH” environmental condition. At the “low temperature, low 

RH” environmental condition, the virus recovery was 131% 

(no outliers were identified as being outside three standard 

deviations of the mean). Recovery greater than 100% may 

be indicative of day-to-day variability within the extraction 

procedure and assay.
12

Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block

Test Duration / 

Sample Inoculum (PFUs)

PFU 

Detectionsa

Recovered Virus 

(PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH

14 Days

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

3.72 x 107

3.72 x 107

0

0

5/5

5/5

0/1

0/1

3.85 ± 0.25 x 107

2.57 ± 0.30 x 107

ND

ND

104 ± 6.66

69.2 ± 7.94

-

-

-

0.2 ± 0.1

-

-

21 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

5.46 ± 5.60 x 100

ND

ND

45.1 ± 19.8

0.00 ± 0.00

-

-

-

5.9 ± 0.4

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

5.81 ± 8.00 x 103

ND

ND

45.1 ± 19.8

0.08 ± 0.11

-

-

-

4.2 ± 1.7

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

3/5

0/1

0/1

3.15 ± 1.38 x 106

4.00 ± 1.82 x 10-1 g

ND

ND

45.1 ± 19.8

0.00 ± 0.00g

-

-

-

6.6 ± 0.1g

-

-

Room Temperature, High RH

1 Day

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

2.55 ± 1.17 x 104

ND

ND

45.1 ± 19.8

0.37 ± 0.17

-

-

-

2.1 ± 0.2

-

-

3 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

3.15 ± 1.38 x 106

NDg

ND

ND

45.1 ± 19.8

0.00 ± 0.00g

-

-

-

6.5 ± 0.0g

-

-

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

3.15 ± 1.38 x 106

NDg

ND

ND

45.1 ± 19.8

0.00 ± 0.00g

-

-

-

6.5 ± 0.0g

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

3.72 x 107

3.72 x 107

0

0

5/5

0/5

0/1

0/1

3.85 ± 0.25 x 107

NDg

ND

ND

104 ± 6.66

0.00 ± 0.00g

-

-

-

7.6 ± 0.0g

-

-
13

Table 2-6. Vaccinia Virus Persistence on Painted Cinder Block (continued)

Test Duration / 

Sample

Inoculum 

(PFUs) PFU Detectionsa

Recovered Virus 

(PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

3.85 ± 0.25 x 107

6.27 ± 3.35 x 107

ND

ND

80.4 ± 5.17

131 ± 70.0

-

-

-

-0.2 ± 0.2

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

3.84 ± 1.73 x 106

ND

ND

45.1 ± 19.8

55.1 ± 24.8

-

-

-

-0.1 ± 0.2

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

3.06 ± 1.25 x 106

ND

ND

45.1 ± 19.8

43.8 ± 18.0

-

-

-

0.0 ± 0.2

-

-

56 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

2.99 ± 2.60 x 104

ND

ND

45.1 ± 19.8

0.43 ± 0.37

-

-

-

2.2 ± 0.4

-

-

Low Temperature, High RH

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

3.85 ± 0.25 x 107

7.68 ± 9.37 x 106

ND

ND

80.4 ± 5.17

16.0 ± 19.6

-

-

-

0.9 ± 0.5

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

2.54 ± 1.60 x 105

ND

ND

45.1 ± 19.8

3.64 ± 2.29

-

-

-

1.2 ± 0.5

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

3.15 ± 1.38 x 106

1.49 ± 1.17 x 105

ND

ND

45.1 ± 19.8

2.13 ± 1.68

-

-

-

1.4 ± 0.3

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

3.15 ± 1.38 x 106

NDg

ND

ND

45.1 ± 19.8

0.00 ± 0.00g

-

-

-

6.5 ± 0.0g

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons. 

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls are inoculated, frozen overnight at ≤-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the 

completion of freeze-drying).

d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.

e

 Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).

f

 Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.

g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for 

non-detects in the calculation of mean log reduction.

h Temperature and RH were actually unknown.

ND = Not detected; the plaque assay detection limit is 10 PFUs.

“-” Not applicable.
14

2.4.4 Industrial Carpet

The vaccinia virus persistence results on industrial carpet are 

summarized in Table 2-7. The vaccinia virus remained viable 

longest under the “low temperature, low RH” environmental 

condition in which ≤4.9 mean log reductions in PFUs were 

observed after 56 days. Under the “room temperature, low 

RH” environmental condition, the vaccinia virus remained 

viable during the 14-day duration with a 0.2 mean log 

reduction in PFUs, but was not detected at ≥21 days. 

Under high RH conditions, the vaccinia virus was not 

detected at room temperature after 1 day, but was viable at 

the low temperature. The vaccinia virus was not detected 

from any of the durations tested (including the shortest 

duration of one day) at the “room temperature, high RH” 

environmental condition. At the “low temperature, high RH” 

environmental condition, the vaccinia virus was detected 

at the 7-, 14-, and 21-day durations with ≤1.5 mean log 

reductions in PFUs; the vaccinia virus was not detected at

42 days.

Table 2-7. Vaccinia Virus Persistence on Industrial Carpet

Test Duration / 

Sample Inoculum (PFUs) PFU Detectionsa

Recovered Virus 

(PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature, Low RH

14 Days

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

3.72 x 107

3.72 x 107

0

0

5/5

5/5

0/1

0/1

6.89 ± 0.61 x 106

4.34 ± 0.34 x 106

ND

ND

18.5 ± 1.65

11.7 ± 0.92

-

-

-

0.2 ± 0.0

-

-

21 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

Room Temperature, High RH

1 Day

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

3.72 x 107

3.72 x 107

0

0

5/5

0/5

0/1

0/1

6.89 ± 0.61 x 106

NDg

ND

ND

18.5 ± 1.65

0.00 ± 0.00g

-

-

-

6.8 ± 0.0g

-

-
15

Table 2-7. Vaccinia Virus Persistence on Industrial Carpet (continued)

Test Duration / Sample Inoculum (PFUs)

PFU 

Detectionsa

Recovered Virus 

(PFUs)b

% Virus 

Recoveryb

Mean Log 

Reductionb

Low Temperature, Low RH

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

6.89 ± 0.61 x 106

1.24 ± 0.51 x 107

ND

ND

14.4 ± 1.28

25.8 ± 10.6

-

-

-

-0.2 ± 0.2

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

1.13 ± 0.27 x 106

9.92 ± 7.96 x 105

ND

ND

16.2 ± 3.81

14.2 ± 11.4

-

-

-

0.2 ± 0.4

-

-

28 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

1.13 ± 0.27 x 106

1.07 ± 0.19 x 105

ND

ND

16.2 ± 3.81

15.4 ± 2.74

-

-

-

0.0 ± 0.1

-

-

56 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

1.13 ± 0.27 x 106

1.55 ± 0.35 x 101

ND

ND

16.2 ± 3.81

0.00 ± 0.00

-

-

-

4.9 ± 0.1

-

-

Low Temperature, High RH

7 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

4.79 x 107

4.79 x 107

0

0

5/5

5/5

0/1

0/1

6.89 ± 0.61 x 106

1.01 ± 0.40 x 106

ND

ND

14.4 ± 1.28

2.11 ± 0.83

-

-

-

0.9 ± 0.2

-

-

14 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

1.13 ± 0.27 x 106

1.32 ± 0.40 x 105

ND

ND

16.2 ± 3.81

1.89 ± 0.57

-

-

-

1.0 ± 0.1

-

-

21 Daysh

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

5/5

0/1

0/1

1.13 ± 0.27 x 106

3.72 ± 2.13 x 104

ND

ND

16.2 ± 3.81

0.53 ± 0.30

-

-

-

1.5 ± 0.2

-

-

42 Days

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

6.98 x 106

6.98 x 106

0

0

5/5

0/5

0/1

0/1

1.13 ± 0.27 x 106

NDg

ND

ND

16.2 ± 3.81

0.00 ± 0.00g

-

-

-

6.1 ± 0.0g

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons. 

b Data are expressed as mean ± standard deviation as applicable. 

c Positive controls are inoculated, frozen overnight at ≤-80°C, freeze-dried until visibly dry (~2 to 4 hours), and then extracted at time zero (at the 

completion of freeze-drying).

d Test coupons are inoculated, freeze-dried, and exposed to the environmental condition for the test duration.

e

 Laboratory blanks are not inoculated with any virus, freeze-dried, and then extracted at time zero (at the completion of freeze-drying).

f

 Procedural blanks are not inoculated with any virus, freeze-dried, and exposed to the environmental condition for the test duration.

g A value of 0 PFUs is used for non-detects in the calculation of recovered virus (PFUs) and % virus recovery, and a value of 1 PFU is used for 

non-detects in the calculation of mean log reduction.

h Temperature and RH were actually unknown.

ND = Not detected; the plaque assay detection limit is 10 PFUs.

“-” Not applicable.
16

2.4.5 Comparison of Persistence Testing Results

The vaccinia virus persistence results are summarized 

in Table 2-8 by denoting the longest duration (days) that 

the virus was detected and the shortest duration that the 

virus was not detected to bracket the length of time that 

the vaccinia virus remained viable after being freezedried on the materials and then held under the various 

environmental conditions. At the “room temperature, low 

RH” environmental condition, the vaccinia virus persisted 

for at least 42 days (the longest duration tested) on glass, 

galvanized metal, and painted cinder block, but was only 

detected after 14 days on industrial carpet. At the “room 

temperature, high RH” environmental condition, the vaccinia 

virus persisted three days or less on all four materials, and 

at the “low temperature, low RH” environmental condition 

the vaccinia virus persisted for at least 56 days (the longest 

duration tested) for all materials. Under the “low temperature, 

high RH” environmental condition, the vaccinia virus 

persisted 14 days on glass, seven days on galvanized metal, 

and 21 days on both painted cinder block and industrial 

carpet. In general, for both the room and low temperature 

tests, the virus remained viable longer at the lower RH.

Table 2-8. Summary of Vaccinia Virus Persistence

Material and Environmental Condition

Longest Duration (Days) Vaccinia 

Virus Detected

Shortest Duration (Days) Vaccinia 

Virus Not Detected

Glass

Room temperature, low RH 42 NA

Room temperature, high RH 3 7

Low temperature, low RH 56 NA

Low temperature, high RH 14 21

Galvanized Metal

Room temperature, low RH 42 NA

Room temperature, high RH 1 3

Low temperature, low RH 56 NA

Low temperature, high RH 7 14

Painted Cinder Block

Room temperature, low RH 42 NA

Room temperature, high RH 1 3

Low temperature, low RH 56 NA

Low temperature, high RH 21 42

Industrial Carpet

Room temperature, low RH 14 21

Room temperature, high RH NA 1

Low temperature, low RH 56 NA

Low temperature, high RH 21 42

NA = Not available; the vaccinia virus was either detected at all durations or not detected from any duration.
17

3.0

Decontamination Technology Evaluation

3.1 Technology Descriptions

The liquid decontamination technologies evaluated

consisted of:

● 1% Citric Acid

○ 1% citric acid was prepared by adding 1 g citric acid, 

anhydrous, (≥ 99.5% purity) to 99 mL hard water until 

completely dissolved.

● 732 ppm Quaternary Ammonium Salt

○ Hospital grade quaternary ammonium disinfectant 

[n-alkyl dimethyl benzyl ammonium chloride (6.25%), 

n-alkyl dimethyl ethylbenzyl ammonium chloride 

(6.25%), inert ingredients (87.5%)] was purchased 

from a local vendor and prepared per the vendor’s 

guidance (3/4 ounces of disinfectant added to 1 gallon 

of hard-water) to obtain a solution containing 732 ppm 

of the quaternary ammonium active ingredient. This 

concentration was not independently verified in this 

evaluation.

All preparations and dilutions were made using Association 

of Official Analytical Chemists (AOAC) hard-water 

prepared at 400 ppm hardness as CaCO3 (AOAC Official 

Method 960.09, Germicidal and Detergent Sanitizing Action 

of Disinfectants, Section E. Synthetic Hard Water, p. 11).

The liquid decontamination technologies were chosen 

for testing because they are common chemicals used for 

disinfection which are readily available. All decontamination 

contact times were 30 minutes.

3.2 Test Matrix

Log reductions in the vaccinia virus were determined for 

two materials (galvanized metal and industrial carpet) 

following 30 minute contact with the liquid decontaminant 

(1% citric acid and 732 ppm quaternary ammonium salt) at 

room temperature (21°C) and low temperature (5°C). The 

experimental treatments performed are shown in Table 3-1. 

The evaluation of liquid decontamination technologies 

utilized test coupons that were each spiked and freeze-dried 

as described in Section 2.2.1 for persistence testing. 

For the decontamination evaluation conducted at the lower 

temperature, the coupons were placed in the refrigerator for 

one hour, before adding the decontamination liquids (which 

were also pre-cooled in the same refrigerator prior to use), 

to equilibrate the materials to the test temperature. In the 

decontamination evaluation spiked test coupons and control 

coupons were inverted (spiked surface down) and placed into 

separate 6 mL troughs each holding enough decontamination 

liquid to cover the spiked surface of the coupon. At the end 

of the 30-minute decontamination contact time, the coupons 

were removed, neutralized with 4.5 mL of Dey and Engley 

(D/E) broth, extracted to recover the vaccinia virus, and 

extracts assayed as described in Section 2.2.2. In addition, 

the trough solution was also analyzed to quantitate any of the 

vaccinia virus that washed into the trough liquid. Since the 

coupons were decontaminated via immersion in a liquid, RH 

was not a relevant parameter to include in the test matrix.

Neutralization approaches for 1% citric acid and 732 ppm 

quaternary ammonium salt followed the methods developed 

from a previous TTEP evaluation of the efficacy of 1% 

citric acid and 732 ppm quaternary ammonium salt against 

the avian influenza H5N1 virus(5). Neutralization consisted 

of 75% D/E broth for both 1% citric acid and 732 ppm 

quaternary ammonium salt (i.e., 3 parts D/E broth to 1 part 

decontamination liquid). During the decontamination 

evaluation with the H5N1 virus, neutralized 1% citric acid 

and 732 ppm quaternary ammonium salt were not cytotoxic 

(>95% cell viability) to Madin-Darby canine kidney 

(MDCK) cells (which were used to quantify the H5N1 

virus)(5). Since MDCK and Vero cells are both mammalian 

kidney epithelial cells, it was not expected (nor subsequently 

observed) that the neutralized 1% citric acid or 732 ppm 

quaternary ammonium salt would induce cytotoxicity in Vero 

Table 3-1. Decontamination Technology Evaluation Matrix

Decontamination Liquid Material Environmental Condition

1% Citric Acid Galvanized metal Room temperature (21°C)

Galvanized metal Low temperature (5°C)

Industrial carpet Room temperature (21°C)

Industrial carpet Low temperature (5°C)

732 ppm Quaternary Ammonium Salt Galvanized metal Room temperature (21°C)

Galvanized metal Low temperature (5°C)

Industrial carpet Room temperature (21°C)

Industrial carpet Low temperature (5°C)
18

cells. In addition, H5N1 virus recoveries from galvanized 

metal and soil exposed to neutralized 1% citric acid and 

732 ppm quaternary ammonium salt were ≥2.60 x 105 tissue 

culture infectious dose of 50%, so an appreciable amount 

of the vaccinia virus was expected to be recovered from 

the neutralized decontamination technologies. In fact, the 

vaccinia virus has been shown to have comparable or less 

susceptibility to antimicrobials than influenza viruses(6-8).

3.3 Technology Evaluation

The vaccinia virus recovery and log reduction in PFUs 

for each decontamination liquid/environmental condition 

combination are summarized in Table 3-2 for galvanized 

metal and Table 3-3 for industrial carpet. Tables 3-2 and 3-3 

also include the number of replicates for positive controls (5) 

test coupons (5), laboratory blanks (1), and procedural 

blanks (1). A summary of the log reductions obtained in all 

decontamination liquid tests is provided in Table 3-4. For the 

decontamination evaluations, the mean room temperature 

was 21°C with a range of 20°C to 22°C and the mean low 

temperature was 5°C with a range of 5°C to 6°C.

None of the decontamination technologies evaluated reduced 

the vaccinia virus on galvanized metal to non-detectable 

levels (Table 3-2). For 1% citric acid, mean log reductions 

in vaccinia virus PFUs on galvanized metal were 3.2 at both 

the room and low temperatures. When 732 ppm quaternary 

ammonium salt was used (at room and the low temperature), 

mean log reductions in vaccinia virus PFUs were 1.5 on 

galvanized metal.

None of the decontamination technologies evaluated reduced 

the vaccinia virus on industrial carpet to non-detectable 

levels (Table 3-3). For 1% citric acid, mean log reductions 

in vaccinia virus PFUs on industrial carpet were 2.6 at room 

temperature and 2.5 at low temperature. When 732 ppm 

quaternary ammonium salt was used (at room and the low 

temperature), mean log reductions in vaccinia virus PFUs on 

industrial carpet were less than 1.0.

Table 3-2. Decontamination Efficacy against Vaccinia Virus on Galvanized Metal

Decontamination Liquid / Sample

Inoculum 

(PFUs)

PFU 

Detectionsa

Mean Recovered 

Virus (PFUs)b

% Virus 

Recoveryb

Mean Log 

Reductionb

Room Temperature

1% Citric Acid

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

2.24 x 107

2.24 x 107

0

0

5/5

5/5

0/1

0/1

2.00 ± 0.47 x 107

1.23 ± 0.23 x 104

ND

ND

89.5 ± 20.9

0.05 ± 0.01

-

-

-

3.2 ± 0.1

-

-

732 ppm Quaternary Ammonium Salt

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.24 x 107

2.24 x 107

0

0

5/5

5/5

0/1

0/1

2.00 ± 0.47 x 107

6.16 ± 1.47 x 105

ND

ND

89.5 ± 20.9

2.75 ± 0.66

-

-

-

1.5 ± 0.1

-

-

Low Temperature

1% Citric Acid

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.60 x 107

2.60 x 107

0

0

5/5

5/5

0/1

0/1

2.09 ± 0.15 x 107

1.37 ± 0.15 x 104

ND

ND

80.53 ± 5.66

0.05 ± 0.01

-

-

-

3.2 ± 0.1

-

-

732 ppm Quaternary Ammonium Salt

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.60 x 107

2.60 x 107

0

0

5/5

5/5

0/1

0/1

2.09 ± 0.15 x 107

7.02 ± 1.10 x 105

ND

ND

80.53 ± 5.66

2.70 ± 0.42

-

-

-

1.5 ± 0.1

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.

b Data are expressed as mean ± standard deviation as applicable.

c Positive controls are virus-inoculated coupons that are freeze-dried, placed into troughs containing PBS (but do not undergo decontamination), 

exposed to the environmental condition, and extracted after the 30-minute contact time. Only one set of positive controls was used for each 

environmental condition and both liquid decontaminants.

d Test coupons are virus-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the 

environmental condition, and extracted after the 30-minute contact time.

e

 Laboratory blanks are non-inoculated coupons that are freeze-dried and extracted at time zero (at the completion of freeze-drying).

f Procedural blanks are non-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the 

environmental condition, and extracted after the 30-minute contact time.

ND = Not detected; the detection limit is 10 PFUs.

“-” Not applicable.
19

Table 3-3. Decontamination Efficacy against Vaccinia Virus on Industrial Carpet

Decontamination 

Liquid / Sample

Inoculum 

(PFUs) PFU Detectionsa

Mean Recovered 

Virus (PFUs)b % Virus Recoveryb

Mean Log 

Reductionb

Room Temperature

1% Citric Acid

Positive Controlc

Test Coupond

Laboratory Blanke

Procedural Blankf

2.24 x 107

2.24 x 107

0

0

5/5

5/5

0/1

0/1

5.62 ± 0.39 x 106

1.43 ± 0.26 x 104

ND

ND

25.1 ± 1.75

0.06 ± 0.01

-

-

-

2.6 ± 0.1

-

-

732 ppm Quaternary 

Ammonium Salt

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.24 x 107

2.24 x 107

0

0

5/5

5/5

0/1

0/1

5.62 ± 0.39 x 106

9.49 ± 5.86 x 105

ND

ND

25.1 ± 1.75

4.24 ± 2.62

-

-

-

0.8 ± 0.2

-

-

Low Temperature

1% Citric Acid

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.60 x 107

2.60 x 107

0

0

5/5

5/5

0/1

0/1

5.15 ± 0.51 x 106

1.69 ± 0.39 x 104

ND

ND

19.82 ± 1.97

0.07 ± 0.02

-

-

-

2.5 ± 0.1

-

-

732 ppm Quaternary 

Ammonium Salt

Positive Control

Test Coupon

Laboratory Blank

Procedural Blank

2.60 x 107

2.60 x 107

0

0

5/5

5/5

0/1

0/1

5.15 ± 0.51 x 106

6.95 ± 0.94 x 105

ND

ND

19.82 ± 1.97

2.67 ± 0.36

-

-

-

0.9 ± 0.1

-

-

a

 PFU detections: the numerator is the number of coupons with PFUs and the denominator is the number of replicate coupons.

b Data are expressed as mean ± standard deviation as applicable.

c Positive controls are virus-inoculated coupons that are freeze-dried, placed into troughs containing PBS (but do not undergo decontamination), 

exposed to the environmental condition, and extracted after the 30-minute contact time. Only one set of positive controls was used for each 

environmental condition and both liquid decontaminants.

d Test coupons are virus-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the 

environmental condition, and extracted after the 30-minute contact time.

e

 Laboratory blanks are non-inoculated coupons that are freeze-dried and extracted at time zero (at the completion of freeze-drying).

f Procedural blanks are non-inoculated coupons that are freeze-dried, placed into troughs containing the liquid decontaminant, exposed to the 

environmental condition, and extracted after the 30-minute contact time.

ND = Not detected; the detection limit is 10 PFUs.

“-” Not applicable.

Table 3-4. Summary of Decontamination Efficacy against Vaccinia Virus

Material and Environmental Condition

Vaccinia Virus Mean Log Reduction in PFUsa

1% Citric Acid 732 ppm Quaternary Ammonium Salt

Galvanized Metal

Room Temperature

Low Temperature

3.2 ± 0.1

3.2 ± 0.1

1.5 ± 0.1

1.5 ± 0.1

Industrial Carpet

Room Temperature

Low Temperature

2.6 ± 0.1

2.5 ± 0.1

0.8 ± 0.2

0.9 ± 0.1

a

 Data are expressed as mean ± standard deviation.
20

This page intentionally blank.
21

4.0

Quality Assurance/Quality Control

Quality assurance/quality control (QC) procedures were 

performed in accordance with the test/QA plan(2) and the 

TTEP QMP(3). QA/QC procedures are summarized below.

4.1 Equipment Calibration

All equipment (e.g., pipettes, incubators, biological safety 

cabinets) used at the time of evaluation was verified as being 

certified, calibrated, or validated.

4.2 Audits

4.2.1 Performance Evaluation Audit

No performance evaluation audit was performed for 

biological agents and organisms because quantitative 

standards for these biological materials do not exist. 

Performance evaluation audits for analytical measurements 

(e.g., temperature, RH, and contact time) were found to be 

acceptable (allowable tolerances are from the test/QA plan(2)), 

as indicated in Table 4-1.

4.2.2 Technical Systems Audit 

Battelle QA staff conducted a technical systems audit (TSA) 

on February 2, April 30, and May 4, 2009 to ensure that 

the evaluation was being conducted in accordance with 

the test/QA plan(2) and the QMP(3). As part of the TSA, test 

procedures were compared to those specified in the test/

QA plan, and data acquisition and handling procedures were 

reviewed. Observations and findings from the TSA were 

documented and submitted to the Battelle Task Order Leader 

for response. No items were noted during the course of the 

TSA with this evaluation. TSA records were permanently 

stored with the TTEP QA Manager.

4.2.3 Data Quality Audit

At least 10% of the data acquired during the persistence 

testing and decontamination technology evaluation were 

audited. A Battelle QA auditor traced the data from 

the initial acquisition, through reduction and statistical 

analysis, to final reporting to ensure the integrity of 

the reported results. All calculations performed on 

the data undergoing the audit were checked.

4.3 QA/QC Reporting 

Each audit (i.e., the performance evaluation audit, TSA, and 

data quality audit) was documented in accordance with the 

QMP(3) for submission to the EPA (the NHSRC QA Manager 

and the Task Order Project Officer). In addition, quality 

control samples including laboratory blanks, procedural 

blanks, and positive controls are reported along with the test 

coupon results in Sections 2 and 3 for each persistence test 

and decontamination evaluation conducted.

4.4 Data Review

Records and data generated from the persistence testing 

and decontamination technology evaluation received a QC/

technical review. All data were recorded by Battelle staff. The 

person performing the QC/technical review was not involved 

in the experiments and added his/her initials and the date to a 

hard copy of the record being reviewed. This hard copy was 

returned to the Battelle staff member who stored the record.

4.5 Deviations

Deviations from the test/QA plan(2) occasionally arose once 

work in the laboratory was initiated. These deviations did 

not adversely affect data quality as described below:

1. Alternative approaches other than those proposed in 

the test/QA plan(2) were used for generating high RH 

and low temperature conditions. Specifically, instead 

of using a nebulizer to generate a high RH at the room 

temperature condition, an RH beyond 70% could be 

maintained by placing 70% humidity beads (Heartfelt 

Industries) inside an airtight Lock&LockTM plastic 

storage container. Therefore the humidity beads were 

used. For reducing temperature, rather than running 

tubing from a re-circulating chiller through the glove 

box, testing was conducted inside a refrigerator. Target 

environmental conditions and acceptable measurement 

tolerances were often not attained with these 

alternative approaches as noted below.

Table 4-1. Performance Evaluation Audit Results

Measurement Audit Procedure Allowable Tolerance Measured Tolerance

Temperature Compare to independent calibrated thermometer value ±2°C ±0.2°C

RH Compare to independent calibrated hygrometer value ±10% ±2%

Contact Time Compare time to independent clock 2 seconds/hour <1 second/hour
22

2. Temperature and RH were found difficult to control 

in some tests and were often outside of the allowable 

test measurement target ranges (±2°C for temperature 

and ±10% for RH) established in the test/QA plan(2), 

as documented in Section 2.3. However, mean 

temperature and RH levels indicate that the test 

conditions of room and low temperatures and low 

and high RH levels were attained. Although some of 

the mean test temperatures deviated from the target 

temperatures by a few °C, the temperature data and 

associated PFU data remain valid and useful. Similarly, 

although there were deviations from the target RH 

levels, the RH and PFU data remain valid.

3. Continuous monitoring of temperature and RH was not 

conducted during the entire vaccinia virus persistence 

duration for the following tests (due to an unplanned 

shut-down/failure of the data loggers):

- The 21-day test at low temperature and low RH 

(monitoring was conducted for less than one day).

- The 28-day test at low temperature and low RH 

(monitoring was conducted for approximately 

15 days).

- The 21-day test at low temperature and high RH 

(monitoring was conducted for approximately 

15 days).

No unusual events occurred that would lead one to 

expect that the temperature and RH of these tests 

deviated from those of comparable tests. However, we 

note that temperature and RH were difficult to control.

4. The target spike amount (inoculum) of the vaccinia 

virus (1 x 107 PFUs per coupon or 1 x 108 PFUs per 

mL) and the associated performance criterion of 

±25% (7.5 x 106 to 1.25 x 107 PFUs per coupon), as 

established in the test/QA plan(2), was sometimes not 

attained during the method demonstration, persistence 

testing, and liquid decontamination evaluation. The 

amount of PFUs in the inoculums (as quantified by 

spike controls and presented as the inoculums for the 

positive and test coupons) ranged from 6.98 x 106 to 

1.67 x 108 PFUs per coupon. These deviations did 

not have any adverse impacts on the testing as an 

appreciable amount of vaccinia virus was spiked to 

quantify log reductions.

5. Method development testing associated with the 

neutralization of the liquid decontaminants was 

not conducted since neutralization methods for the 

liquid decontaminants tested (1% citric acid and 732 

ppm quaternary ammonium salt) were previously 

established in the companion TTEP evaluation of 

liquid decontamination technologies for the H5N1 

virus(5).

6. Regarding the decontamination technology evaluation, 

the positive controls (at the 30-minute contact time) 

were exposed to PBS rather than being exposed only to 

air, which better mimicked the actual application of the 

decontamination liquid. 
23

5.0

Summary

5.1 Vaccinia Virus Persistence

The persistence of the vaccinia virus was investigated on four 

materials (glass, galvanized metal, painted cinder block, and 

industrial carpet), after being inoculated onto the materials 

and freeze-dried, then held under four environmental 

conditions (comprised of room and low temperatures and 

low and high RH) for various durations. Under low RH 

conditions at both room and low temperature, the vaccinia 

virus persisted at least for 14 days on all materials. The 

vaccinia virus persisted for 42 days on glass, galvanized 

metal, and painted cinder block at the “room temperature, 

low RH” environmental condition, and the vaccinia virus 

persisted for 56 days on all four materials at the “low 

temperature, low RH” environmental condition.

The persistence of the vaccinia virus was generally reduced 

under high RH conditions at both room and low temperatures. 

Under the “room temperature, high RH” environmental 

condition, the vaccinia virus remained viable on glass for 

three days and remained viable on galvanized metal and 

painted cinder block for one day; the vaccinia virus was not 

viable on industrial carpet at the one-day duration. Under 

the “low temperature, high RH” environmental conditions, 

the vaccinia virus was detected on glass after 14 days, 

detected on galvanized metal after seven days, and detected 

on painted cinder block and industrial carpet after 21 days.

5.2 Vaccinia Virus Liquid Decontamination 

The efficacy of two liquid decontamination technologies 

(1% citric acid and hospital grade quaternary ammonium 

salt disinfectant at 732 ppm) was evaluated for the vaccinia 

virus inoculated and freeze-dried on galvanized metal and 

industrial carpet. Decontamination efficacies were assessed 

at room and low temperatures with a 30-minute liquid 

decontaminant contact time.

Neither liquid decontamination technology reduced the 

vaccinia virus to non-detectable levels. For 1% citric acid, 

mean log reductions in vaccinia virus on galvanized metal 

were 3.2 at both the room and low temperatures; on industrial 

carpet log reductions were 2.6 at room temperature and 

2.5 at the low temperature. When 732 ppm quaternary 

ammonium salt was used, mean log reductions in vaccinia 

virus on galvanized metal were 1.5 at both the room and 

low temperature conditions; on industrial carpet the log 

reductions were less than 1.0.
24

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25

6.0

References

1. Fulginiti,V.A., A. Papier, J.M. Lane, J.M. Neff, and D.A. Henderson. 2003. Smallpox Vaccination: A Review, Part I. 

Background, Vaccination Technique, Normal Vaccination and Revaccination, and Expected Normal Reactions. Clinical 

Infectious Diseases, 37:241–250. http://www.journals.uchicago.edu/doi/pdf/10.1086/375824.

2. Technology Testing and Evaluation Program Test/QA Plan for Persistence Testing of Avian Influenza Viruses on Outdoor 

Materials, Version 1, Battelle, Columbus, Ohio, March 2007.

3. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP), Version 1, Battelle, 

Columbus, Ohio, January 2005.

4. Stone, H.J., J.V. Rogers, E.J. Fleming, Y.W. Choi, J.D. Waugh, W.R. Richter, M.L. Taylor, K.B. Riggs, Z.J. Willenberg, R.T. 

Krile, and S. Ryan. 2006. Investigation Report: Impact of Temperature and Humidity on the Persistence of Vaccinia Virus 

and Ricin Toxin on Indoor Surfaces. EPA/600/R-08/002.

5. Choi, Y.W., J.V. Rogers, D.J. Chappie, M.L. Taylor, K.B. Riggs, Z.J. Willenberg, and J.P. Wood. 2009. Investigation and 

Technology Evaluation Report: Highly Pathogenic Avian Influenza H5N1 Virus Persistence Testing and Evaluation of 

Liquid Decontamination Technologies. EPA/600/R-09/054.

6. Hamouda, T., A. Myc, B. Donovan, A.Y. Shih, J.D. Reuter, and J.R. Baker, Jr. 2001. A Novel Surfactant Nanoemulsion 

with a Unique Non-Irritant Topical Antimicrobial Activity Against Bacteria, Enveloped Viruses and Fungi. Microbiological 

Research, 156:1–7. 

7. Sugimoto, Y. and S. Toyoshima. 1979. Nα-Cocoyl-L-Arginine Ethyl Ester, DL-Pyroglutamic Acid Salt, As an Inactivator of 

Hepatitis B Surface Antigen. Antimicrobial Agents and Chemotherapy, 16:329–332.

8. Kampf, G., J. Steinmann, and H. Rabenau. 2007. Suitability of Vaccinia Virus and Bovine Viral Diarrhea Virus (BVDV) for 

Determining Activities of Three Commonly-Used Alcohol-Based Hand Rubs Against Enveloped Viruses. BMC Infectious 

Diseases, 7:5. 
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