&EPA
United States
Environmental Protection
Agency
Highly Pathogenic Avian Influenza
H5N1 Virus Persistence Testing
and Evaluation of Liquid I
Decontamination Technologies
INVESTIGATION AND TECHNOLOGY
EVALUATION REPORT
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-09/054 | October 2009 www.epa.gov/ord
INVESTIGATION AND TECHNOLOGY
EVALUATION REPORT
Highly Pathogenic Avian Influenza
H5N1 Virus Persistence Testing and
Evaluation of Liquid Decontamination
Technologies
By
Young W. Choi, 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
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Disclaimer
The U.S. Environmental Protection Agency, through its Office of Research and
Development's National Homeland Security Research Center, funded, directed, and
managed this investigation and technology evaluation through a Blanket Purchase
Agreement under General Services Administration contract number GS23F0011L-3
with Battelle. All tests were conducted at Battelle facilities located in West Jefferson,
Ohio. It has been reviewed by U.S. Environmental Protection Agency but does not
necessarily reflect the Agency's views. No official endorsement should be inferred.
U.S. Environmental Protection Agency does not endorse the purchase or sale of any
commercial products or services.
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Foreword
The U.S. Environmental Protection Agency is charged by
Congress with protecting the nation's air, water, and land
resources. Under a mandate of national environmental
laws, the U.S. Environmental Protection Agency 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
Agency's Office of Research and Development 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, the Agency announced the formation
of the National Homeland Security Research Center. The
Center is part of the Office of Research and Development;
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 Center has developed the Technology Testing and
Evaluation Program in an effort to provide reliable
information regarding the performance of homeland security
related technologies. The Technology Testing and Evaluation
Program 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, and for
decontaminating structures and the outdoor environment.
The evaluation reported herein was conducted by Battelle
as part of Technology Testing and Evaluation Program.
Information on National Homeland Security Research Center
and Technology Testing and Evaluation Program can be
found at http://www.epa.gov/nhsrc.
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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
the following U.S. Environmental Protection Agency's personnel for their reviews Dr.
Worth Calfee (National Homeland Security Research Center); Dr. Wendy Davis-Hoover
(National Risk Management Research Laboratory); and Mr. Jeff Kempter (Office of
Pesticide Programs).
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Contents
Disclaimer iv
Foreword v
Acknowledgments vi
Abbreviations/Acronyms ix
Unit Abbreviations x
Definitions x
Executive Summary xi
1.0 Introduction 1
2.0 Persistence Testing 3
2.1 Test Materials 3
2.2 Cytotoxicity of Material Extracts 4
2.3 H5N1 Virus Recovery From Test Materials 4
2.3.1 Spiking the Coupons 4
2.3.2H5N1 Virus Extraction and Quantification 5
2.3.3 H5N1 Virus Recoveries Based onResults of Virus Spiking Studies 6
2.4 Persistence Testing Approach 10
2.4.1 Temperature 10
2.4.2 Relative Humidity 11
2.4.3 Ultraviolet-A/B Radiation 12
2.5 Test Results 13
2.5.1 Chicken Feces 13
2.5.2 Galvanized Metal 16
2.5.3 Glass 18
2.5.4 Soil 20
2.5.5 Comparison of Chicken Feces, Galvanized Metal, Glass, and Soil Results 22
3.0 Decontamination Technology Evaluation 25
3.1 Technology Descriptions 25
3.2 Cytotoxicity of Neutralized Decontamination Liquids 25
3.3 H5N1 Virus Recovery From Extraction Buffer, Neutralized Decontamination Liquid,
and Trough Decontamination Liquid 26
3.3.1 H5N1 Virus Recovery From Coupons Using Extraction Buffer 26
3.3.2 H5N1 Virus Recovery From Neutralized Decontamination Liquid 27
3.3.3 H5N1 Virus Recovery From Trough Decontamination Liquid 27
3.4 Evaluation of Liquid Decontamination Technologies 28
3.4.1 Test Matrix 28
3.4.2 Test Results 28
4.0 Quality Assurance/Quality Control 33
4.1 Equipment Calibration 33
4.2 Audits 33
4.2.1 Performance Evaluation Audit 33
4.2.2 Technical Systems Audit 33
4.2.3 Data Quality Audit 33
4.3 Quality Assurance/Quality Control Reporting 33
4.4 Data Review 33
4.5 Deviations 33
5.0 Summary 35
5.1 H5N1 Virus Persistence 35
5.2 H5N1 Virus Liquid Decontamination 35
6.0 References 37
Appendix A: H7N2 Virus Testing 39
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Figures
Figure 2-1. Petri Dishes With Soil 5
Figure 2-2. Soil and Parafilm® in a Conical Vial 6
Figure 2-3. Mini-Refrigerator Configured With Ultraviolet A/B Lamps 12
Figure 2-4. Schematic (Top View) of Ultraviolet Sampling Locations 13
Tables
Table ES-1. Summary of Persistence Test Conditions and H5N1 Virus Recoveries xii
Table 2-1. Test Materials 3
Table 2-2. Material Extract Cytotoxicity Test Results 4
Table 2-3. H5N1 Virus Recovery FromBasswood 7
Table 2-4. H5N1 Virus Recovery From Chicken Feces 7
Table 2-5. H5N1 Virus Recovery From Concrete (Unpainted) 8
Table 2-6. H5N1 Virus Recovery From Galvanized Metal 8
Table 2-7. H5N1 Virus Recovery From Glass 9
Table 2-8. H5N1 Virus Recovery From Pine Wood 9
Table 2-9. H5N1 Virus Recovery From Soil 9
Table 2-10. Persistence Test Matrix 11
Table 2-11. Ultraviolet Measurements 13
Table 2-12. H5N1 Virus Persistence on Chicken Feces 14
Table 2-12. H5N1 Virus Persistence on ChickenFeces (continued) 15
Table 2-13. H5N1 Virus Persistence on Galvanized Metal 16
Table 2-13. H5N1 Virus Persistence on Galvanized Metal (continued) 17
Table 2-14. H5N1 Virus Persistence on Glass 18
Table 2-14. H5N1 Virus Persistence on Glass (continued) 19
Table 2-15. H5N1 Virus Persistence on Soil 20
Table 2-15. H5N1 Virus Persistence on Soil (continued) 21
Table 2-16. Summary of H5N1 Virus Persistence 22
Table 3-1. Neutralized Decontamination Liquid Cytotoxicity Test Results 25
Table 3-2. H5N1 Virus Recovery From Coupons Using Extraction Buffer 26
Table 3-3. H5N1 Virus Recovery From Neutralized Decontamination Liquid 27
Table 3-4. H5N1 Virus Recovery From Galvanized Metal Trough Decontamination Liquid 28
Table 3-5. Decontamination Technology Evaluation Matrix 28
Table 3-6. Decontamination Efficacy Against H5N1 Virus on Galvanized Metal 29
Table 3-7. Decontamination Efficacy Against H5N1 Virus on Soil 30
Table 3-8. Summary of Decontamination Efficacy Against H5N1 Virus 31
Table A-l. H7N2 Virus Titers Obtained Using Chicken Embryo Kidney Cells 39
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Abbreviations/Acronyms
AOAC AOAC International (formerly the Association of Analytical Chemists)
°C degrees Celsius
CaCO3 calcium carbonate
CEK chicken embryo kidney
CO2 carbon dioxide
CPE cytopathic effects
D/E Dey and Engley
EPA U.S. Environmental Protection Agency
H5N1 highly pathogenic avian influenza virus
H7N2 low pathogenic avian influenza virus
MDCK Madin-Darby canine kidney
MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
Na2CO3 sodium carbonate
NHSRC National Homeland Security Research Center
PBS phosphate-buffered saline
QA quality assurance
QC quality control
QMP quality management plan
RH relative humidity
TCID50 50% tissue culture infectious dose
TTEP Technology Testing and Evaluation Program
UV ultraviolet radiation
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Unit Abbreviations
°c
cm
g
hr
min
mL
mm
ppm
rpm
uL
um
uw
degrees Celsius
centimeter
gram
hour
minutes
milliliter
millimeter
parts per million
revolutions per minute
microliter
micrometer
microwatt
Definitions
TCID50 is a quantitative measure of infectivity and growth and is the number of
organisms that must be present to infect 50% of the cell culture wells tested.
UV-A/B ultraviolet radiation within the wavelength range of 280-400 nanometers
UV-A 320-400 nanometers
UV-B 280-320 nanometers
UV-C ultraviolet radiation within the wavelength range of 200-280 nanometers
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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 by carrying
out performance tests on homeland security technologies.
Under TTEP, the persistence of the highly pathogenic avian
influenza H5N1 virus on test coupons prepared from chicken
feces, galvanized metal, glass, and soil was investigated.
The performance of liquid technologies to decontaminate the
H5N1 virus from test coupons of galvanized metal and soil
was also investigated at room and low temperatures.
For persistence testing, test coupons were contaminated
by spiking each coupon with a quantity of H5N1 virus
amounting to a 50% tissue culture infectious dose (TCID50)
of H5N1 virus (A/Vietnam/1203/04) of at least 1 x 106. The
persistence of the H5N1 virus was investigated for up to
three test durations at five different environmental conditions
[each environmental condition is described by a specific
temperature, relative humidity (RH) value, and presence/
absence of exposure to ultraviolet (UV-A/B) radiation to
simulate sunlight]. Please note that all references to and
quantifications of the H5N1 virus in this document refer to
viable H5N1 virus. The environmental conditions were:
• Room temperature, low RH, no UV
° Test durations: 1-, 2-, and 4-day
• Room temperature, high RH, no UV
° Test durations: 1-, 2-, and 4-day
• Low temperature, low RH, no UV
° Test durations: 4-, 8-, and 13-day
• Low temperature, high RH, no UV
° Test durations: 4-, 9-, and 13-day
• Low temperature, low RH, UV-A/B
° Test durations: 24-, 48-, and 96-hours
(continuous UV-A/B exposure)
Note that for all experiments, the target room temperature
was 22 degrees Celsius (°C), target low temperature was
4 °C, target high RH was 80%, and target low RH was 40%.
The persistence testing yielded the following results. At room
temperature (under low RH and high RH, with no UV), the
H5N1 virus did not persist on galvanized metal and glass at
time periods of one day or greater while generally persisting
on chicken feces and soil for less than two days.
At low temperature (under low RH and high RH, with no
UV) the H5N1 virus persisted for at least four days on all
materials. Following exposure to the low temperature, low
RH, no UV environmental condition, the H5N1 virus was
detected after 13 days on galvanized metal, glass, and soil.
The H5N1 was also viable following exposure to the low
temperature, high RH, no UV environmental condition,
after nine days on chicken feces, glass, and soil. Although
testing was not conducted for durations longer than 13 days
for any of the environmental conditions, the H5N1 virus
persistence may exceed 13 days, especially on galvanized
metal and glass under the low temperature, low RH, no UV
environmental condition.
With continuous UV-A/B exposure (and under a low
temperature and low RH environmental condition), the H5N1
virus persisted less than 48 hours on galvanized metal and
glass but persisted at least 48 hours on chicken feces and soil.
A summary of the actual test conditions and the quantities
of the H5N1 virus (expressed as individual TCID50 values)
recovered from chicken feces, galvanized metal, glass, and
soil is provided in Table ES-1.
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Table ES-1. Summary of Persistence Test Conditions and H5N1 Virus Recoveries3
!
Room Temperature,
LowRH, NoUV
i P 1 1 1 1 1 1 Jllplflpp
li^ii! 1111 Illfe^;^**)* i^i^!)**S!)!iil!l!l!l!l^
1-Day 23 35 6.37xl02c ND ND 8.82xl03d
2-Day 22 36 ND ND ND ND
4-Day 23 49 ND ND ND ND
Room Temperature,
High RH, NoUV
1-Day 22 89 7.70xl04 ND ND 8.26xl04
2-Day 23 90 3.16xl03c ND ND ND
4-Day 24 91 ND ND ND ND
Low Temperature,
LowRH, NoUV
4-Day 4 28 7.11xl04 3.78xl05 4.98xl05 2.47xl04
8-Day 7 15 3.16xl03c 7.33 x 105 1.34xl06 1.16xl03
13-Day 7 46 ND 3.44x10= 5.91 x 105 1.05xl03d
Low Temperature,
High RH, No UV
4-Day 8 89 5.42 x 104 4.69xl03 1.37 x 105 2.21 x 105
9-Day 7 97 5.90 x 104 ND 3.16xl03 5.93x10=
13-Day 7 79 7.86xl02d ND ND ND
Low Temperature,
LowRH, UV-A/B6
24-Hourse 4 25 1.06xl06 6.42 x 104 2.62 x
102d 5.93xl05
48-Hourse -1 30 9.56 x 104 ND ND 3.90xl04
96-Hourse 0.1 28 3.16xl03c Not Tested Not Tested 3.11xl03d
a Spike amount ranged from 5.01 x 106 to 5.01 x 107 TCID50.
b Mean temperature and RH values were based on continuous monitoring at 1-minute intervals, with the exception of the room temperature, low
RH, no UV environmental condition, which were derived from the mean of the temperature or RH at the start and end of the tests.
0 TCID50 was detected but at a level < the procedural blank TCID50 for chicken feces such that the cytopathic effects observed (and used in the
calculation of the TCID50) may be attributed to the test material rather than H5N1 virus.
d H5N1 virus was not detected on some of the replicate test coupons; a value of 1 TCID50 was used for non-detects in the calculation of the mean
H5N1 virus recovery.
e UV-A/B exposures were continuous (i.e., the UV-A/B lamps did not shut off every 12 hours).
ND = No cytopathic effects detected; the detection limit was 1.31 x 103 TCID50.
The liquid decontamination technologies evaluated
included 1% citric acid, pH-amended bleach, a hospital
grade 732 parts per million (ppm) quaternary ammonium
disinfectant, and 8% sodium carbonate (Na2CO3) (all were
prepared in water with a hardness of 400 ppm as calcium
carbonate [CaCO3]). The H5N1 virus was inoculated onto
galvanized metal and soil test coupons and exposed to the
decontamination liquid for a 10-minute contact time. The
decontamination technology evaluation was implemented at
room temperature and at a low temperature; UV-A/B lamps
were not used.
The decontamination technology evaluation results indicated
that only pH-amended bleach was completely effective
at inactivating the H5N1 virus (i.e., H5N1 virus was not
detected), and this inactivation only occurred on galvanized
metal (at both room and low temperatures). On soil, pH-
amended bleach induced mean H5N1 virus log reductions
in TCID50 of 2.7 at room temperature and 2.9 at low
temperature. The mean H5N1 virus log reductions in TCID50
associated with 1% citric acid ranged from 1.5 (with soil
at room temperature) to 2.1 (with galvanized metal at low
temperature). The mean H5N1 virus log reductions in TCID50
associated with 732 ppm quaternary ammonium and 8%
Na2CO3 were less than 1.0.
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1.0
Introduction
National Homeland Security Research Center's (NHSRC's)
Technology Testing and Evaluation Program (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, the persistence of viable, highly pathogenic
avian influenza H5N1 virus was tested and the performance
of liquid decontamination technologies to inactivate the
H5N1 virus was evaluated. The primary objectives were
to determine how long and under what environmental
conditions the H5N1 virus remains viable; and to evaluate the
efficacy of four generic liquid decontamination technologies.
Persistence testing investigated the amount of the H5N1 virus
remaining on chicken feces, galvanized metal, glass, and soil
for one to three different durations under the following five
environmental conditions:
• Room temperature, low relative humidity (RH), no
ultraviolet radiation (UV)
• Room temperature, high RH, no UV
• Low temperature, low RH, no UV
• Low temperature, high RH, no UV
• Low temperature, low RH, UV-A/B
Note that the target room temperature was 22 degrees Celsius
(°C), the target low temperature was 4 °C, the target high RH
was 80%, and target low RH was 40%.
Four liquid decontamination technologies (1% citric acid,
pH-amended bleach, 732 parts per million (ppm) quaternary
ammonium and 8% sodium carbonate [Na2CO3]) prepared
in hard water were evaluated for their effectiveness in
inactivating the H5N1 virus. For this technology evaluation,
the H5N1 virus was spiked onto two materials (galvanized
metal and soil) and the spiked test coupons were treated
with the liquid decontaminant for a 10-minute contact
time at room temperature and a low temperature (UV-A/B
lamps were not used for the decontamination technology
evaluation).
Efforts were conducted according to a peer-reviewed test/
QA plan1 that was developed according to the requirements
of the TTEP quality management plan (QMP)2. The
persistence testing and the technology evaluation both used
a TCID50 (50% tissue culture infectious dose) assay to
quantify the H5N1 virus extracted from test coupons. This
report documents the log reductions in TCID50 associated
with a natural reduction in the H5N1 virus under various
environmental conditions and the decrease in the H5N1 virus
exposed to various liquid decontamination technologies.
Please note that the test/QA plan1 also includes testing for
the low pathogenic avian influenza virus H7N2. Although
persistence testing and evaluation of liquid decontamination
technologies were not conducted for the H7N2 virus,
preliminary research results associated with the H7N2 virus
are included in Appendix A.
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2.0
Persistence Testing
2.1 Test Materials
Materials considered for H5N1 virus persistence testing
are described in Table 2-1. Basswood, concrete, and pine
wood were eventually rejected for H5N1 virus persistence
testing because of the poor recovery of the H5N1 virus
(i.e., <5% of the applied inoculum of approximately 1 x 106
TCID50 of H5N1 virus per coupon) from these materials
(see Section 2.3). Persistence testing was performed using
test coupons of uniform size. As indicated in Table 2-1 test
coupons for wood, galvanized metal, and glass were each
1.9 centimeter (cm) x 7.5 cm; concrete test coupons were
each 1.0 cm x 3.5 cm. Chicken feces and soil test coupons
consisted of 3.5 cm diameter Petri dishes, 1 cm high, lined
with Parafilm®, and filled with uncompacted material. Straw
and feathers were removed from the chicken feces before
being placed in the Petri dishes.
Table 2-1. Test Materials
illllllllllbM^fes^
rfSfe ^^^^^^^^^^^^^^^^^^ $aW$&
Basswood (a hardwood) Not applicable
Chicken Feces Not applicable
Concrete (unpainted) 5 parts sand: 2 parts
cement
BSBs^-y'ypJispfs^!!?^;
i^^M^i^UilJ^kik^v^
•i\^^^y^'^>i^^:'i^^^^^''^^
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2.2 Cytotoxicity of Material Extracts
Prior to persistence testing and the decontamination
technology evaluations, the potential for extracts of test
coupons (with no virus present) to cause cytotoxicity
was determined. Cytotoxic effects caused by test coupon
extracts could interfere with the TCID50 determination.
The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assay was used to determine the cytotoxic
effects of test coupon extracts. For the MTT assay, Madin-
Darby canine kidney (MDCK) cells (the cells used to
quantitate the TCID50) were seeded into each well of a
96-well microtiter plate and exposed to serial dilutions of
test material extracts. MDCK cells exposed only to serial
dilutions of sterile cell culture medium (the medium in which
the virus particles were suspended) were used as controls.
The MDCK cells were then incubated in the presence of
MTT where mitochondrial succinate dehydrogenase (present
in living cells) converts the yellow MTT to a purple formazan
salt. The absorbance (optical density) of this purple reaction
product is determined using a microplate reader and is
proportional to mitochondrial function (i.e., the number of
living cells) and is expressed as a percentage of the control
cell viability.
The cytotoxicity results showing the lowest dilution able to
achieve >90% of the control cell viability are summarized in
Table 2-2. The cell viability performance criterion (per the
test/QA plan1) of >90% of unexposed control viability with
dilution of 1 to 8 (1:8) or less was attained for all materials
except soil. However >90% of unexposed soil control
viability was obtained with a dilution of 1:16, which was
approved as an acceptable level of dilution by the EPA Task
Order Project Officer. Although an increased level of dilution
was needed for the MTT assay with soil, the TCID50 assay
was not believed to be affected. This is because cytopathic
effects were not generally observed in the MDCK cells
exposed to any dilution of non-inoculated soil extract; the
detection limit of the TCID50 assay was not generally affected
by soil. Further, any potential negative bias in the TCID50
results due to cytotoxicity of the soil extract would have been
indicated through the inadequate positive control recovery,
which was not the case.
2.3 H5N1 Virus Recovery From Test Materials
2.3.1 Spiking the Coupons
In order to assess the quantity of virus that could be
recovered, following the addition of a known quantity of
virus to each of several test coupons, each coupon was
spiked with a quantity of virus ranging from 1.98 x 106 to
7.93 x 106 TCID50 of H5N1 virus per coupon. One hundred
microliters [uL] of stock virus suspension was applied per
coupon; the target spike level was 1 x 106 TCID50 of H5N1
virus per coupon. The H5N1 virus was a Battelle stock
propagated from a parent stock provided by the Centers for
Disease Control and Prevention. This H5N1 influenza A
virus is the 1,203rd strain isolated from a human in Vietnam
in 2004 (A/Vietnam/1203/04). Genetic sequencing was used
to verify that propagated stocks matched the parent stock.
Spiking of the 1.9 cm x 7.5 cm coupons was conducted using
a multichannel micropipette as two rows of five droplets
(10 uL per droplet) across the surface of the test coupon. A
single channel pipette was used to apply ten 10 uL droplets
at separate locations on the surface of each chicken feces and
soil test coupon. For concrete, which readily absorbs liquid,
the spiking was conducted using a single channel pipette and
100 uL of the inoculum was applied as a single line or streak
across the surface of the test coupon.
After spiking, all coupons were allowed to dry undisturbed at
22 ± 2 °C and 40-70% RH for one hour prior to H5N1 virus
extraction. Test coupons were placed in an acrylic Compact
Glove Box Model 830-ABC (Plas Labs, Inc., Lansing,
Mich.), with a volume of 317 liters. Feces collected from
chickens on the morning of testing remained moist during
the one-hour drying time. The soil, stored in an air-tight
container prior to use, also remained moist during the one-
hour drying time. Following the one-hour drying time, the
H5N1 virus inoculum appeared to absorb into chicken feces
and soil and was not visible; however, spots indicative of the
dried H5N1 virus inoculum were visible on galvanized metal
and glass.
Table 2-2. Material Extract Cytotoxicity Test Results
Bass wood
0.3612
1:2
0.4604
127b
Chicken Feces
0.3108
1:4
0.4114
132b
Concrete
0.5557
1:8
0.5075
91.3
Glassc
Pine Woodd
Not Tested
Not Tested
Not Tested
Not Tested
Galvanized Metal
0.2389
1:8
0.2216
Soil
0.5657
1:16
0.5255
92.9
a The optical density (absorbance) is directly proportional to the number of viable cells present in the sample well.
b Viability above 100% may reflect non-specific reduction of MTT to formazan from unknown coupon extract components.
c The cytotoxicity associated with glass was not evaluated as previous testing indicated there is no interference with MDCK cell viability.
d Pine wood was not tested due to insufficient recovery of the H5N1 virus; see section 2.3.3.
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2.3.2 H5N1 Virus Extraction and Quantification
For basswood, concrete, galvanized metal, glass, and
pine wood, extraction of the H5N1 virus from individual
test coupons entailed, following drying, placing a single
spiked test coupon into a sterile 50 milliliter (mL) conical
vial containing 10 mL of sterile extraction buffer (i.e.,
phosphate-buffered saline [PBS]). The vials were agitated on
an orbital shaker for at room temperature for 15 minutes at
approximately 200 revolutions per minute (rpm). The extract
was removed from the vial using a pipette and was then
serially diluted and plated onto MDCK cells. Quantitation
of the H5N1 virus in the extract was based on 10-fold serial
dilutions of the material extracts applied to MDCK cells and
the subsequent observation of cytopathic effects (CPE) in
the MDCK cells to determine the viral concentration. The
quantity of virus present was expressed as the TCID50 for the
H5N1 virus.
The TCID50 for H5N1 virus in test coupon extracts was
determined by first preparing 10-fold serial dilutions of each
extract and then transferring 0.1 mL aliquots of each dilution
to five wells (of a 96-well microtiter plate) containing
mono layers of MDCK cells. Following transfer of aliquots
of the diluted extracts, the 96-well microtiter plate was
incubated at 37 ± 2 °C under 5% carbon dioxide (CO2) in a
humidified incubator for 72 to 96 hours or until CPE were
visually detected by comparing to the control (cell culture
medium only) wells. Using light microscopy each well of the
96-well plate was evaluated and observations documented
(+ = positive CPE; 0 = no observed effect) for CPE. The
TdD50 was calculated using the Spearman-Karber3 method
as follows:
Equation 2-1.
Unit Volume
(Highest Dilution^
with 100% CPE }'
Cumulative Number of Wells
with CPE at that Dilution
Number of Wells per Dilution
For the liter, take the inverse log of TCID50 per unit volume.
For an inoculating volume of 0.1 mL, the calculated TCID50
titer is multiplied by a factor of 10 (10 x 0.1 mL =1.0 mL) to
achieve TCID50/mL.
The TCID50 assay's limit of quantitation (i.e., detection limit)
for all materials using the Spearman-Karber method3'4 is 131
TCID50/mL (i.e., 1.31 x 103 TCID50 per coupon).
For quantitating the H5N1 virus in chicken feces and soil,
each test coupon plus the Parafilm®, which lined each Petri
dish (Figure 2-1), were placed in the conical vials containing
the PBS extraction buffer. (Figure 2-2). Each vial was shaken
on the orbital shaker as described for the other test materials
(shaken on an orbital shaker at 200 rpm for 15 minutes),
then each vial was centrifuged at 1000 xg for 10 minutes.
A syringe was used to withdraw a 0.5 to 1 mL aliquot of the
extract in each vial, which was then filtered using a 0.2 um
(polyether sulfone, low protein-binding) syringe filter,
enabling the H5N1 virus to pass through while excluding
larger particles and organisms. The filtered sample was then
serially diluted and plated onto the MDCK cells for TCID50
determination.
Figure 2-1. Petri Dishes With Soil
-------
Figure 2-2. Soil and Parafilm® in a Conical Vial
2.3.3 H5N1 Virus Recoveries Based on Results of Virus
Spiking Studies
Materials considered for persistence testing were studied to
determine if an acceptable level of H5N1 virus (an acceptable
level is defined in the test/QAplan1 as >5% of the applied
inoculum) could be recovered from the coupons. The
coupons were spiked and allowed to dry for one hour; then
the H5N1 virus was extracted from the coupons and analyzed
for TCID50 determinations. Numerous extraction approaches
and materials (e.g., the use of surfactants) were considered
for use in an attempt to achieve acceptable H5N1 virus
recovery. Initial determinations of H5N1 virus recoveries
were conducted with three replicate coupons per material;
if acceptable recoveries were obtained final demonstrations
using five replicate coupons were used to obtain mean
recovery and variance data. The H5N1 virus recovery results
and associated extraction approaches for each of the materials
tested are provided in Tables 2-3 through 2-9. Materials
studied include: basswood (a hardwood), chicken feces,
concrete, galvanized metal, glass, pine wood (a softwood),
and soil. For concrete, special tests were also conducted
to investigate the impact of the highly alkaline concrete
extraction solution on the viability of the H5N1 virus and the
adsorption/deposition of the H5N1 virus into the concrete
(Table 2-5). The tabulated data indicate that an acceptable
H5N1 virus recovery (>5% of the applied inoculum) was
only obtained from chicken feces, galvanized metal, glass,
and soil. Hence, these four materials were used in subsequent
persistence testing.
-------
Table 2-3. H5N1 Virus Recovery From Basswood
Basswood; 0.7 cm thickness
Placed in PBS, agitated on an
orbital shaker for 15 minutes (min)
at 200 rpm.
2.00xl06 5.01 ±0.00 xlO3 0.25 ± 0.00
Basswood; 0.2 cm thickness
Placed in PBS, agitated on an
orbital shaker for 15 min at 200
rpm.
2.00xl05 3.00± 1.74xl03 0.15 ±0.09
Basswood; 0.2 cm thickness
Basswood soaked in 2% skim milk
(dry milk reconstituted in deionized
water) for 10 min, allowed to dry
to the touch, and autoclaved for
15 min at 121 °C. Once cooled,
inoculated and allowed to dry for
1 hour (hr), then placed in PBS,
agitated on an orbital shaker for
15 min at 200 rpm, coupons were
also scraped with a pipette tip,
and extraction fluid jetted onto the
inoculated surface in an attempt to
aid H5N1 virus recovery.
2.00xl06 4.75 ±2.76 x 103 0.24 ±0.14
Basswood; 0.2 cm thickness
Placed in PBS, agitated on a plate
shaker for 5 min at 1,100 rpm.
5.01xl06 3.00± 1.73x 103 0.06 ± 0.03
Basswood; 0.2 cm thickness
Placed in PBS, agitated on a
vortexer for 2 min at 2,500 rpm.
5.01xl06 4.00± 1.73x 103 0.08 ± 0.03
' Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
Table 2-4. H5N1 Virus Recovery From Chicken Feces
Chicken Feces Placed in PBS, agitated on an orbital
shaker for 15 min at 200 rpm, then
centrifuged with the supernatant filtered
(0.2 urn) for analysis.
2.00x 106
1.74± 1.25xl05
8.7 ±6.3
Chicken Feces Placed in PBS, agitated on an orbital
shaker for 15 min at 200 rpm, then
centrifuged with the supernatant filtered
(0.2 urn) for analysis.
7.92x 106
5.23 ± 1.72x10=
6.6 ±2.2
' Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
-------
Table 2-5. H5N1 Virus Recovery From Concrete (Unpainted)
Concrete
Placed in PBS, agitated on an orbital shaker
for 15 min at 200 rpm.
1.98xl06
ND
0.00 ±0.00
Concrete; The PBS extraction of concrete resulted in a
extract highly alkaline solution (i.e., pH > 11). A test
solution was conducted to determine if this solution
inactivates the H5N1 virus. Concrete was
placed in PBS and shaken to obtain a high
alkaline solution, the coupon was removed
and the solution was spiked with H5N1 virus
then agitated on an orbital shaker for 15 min
at 200 rpm.
5.01 x 106 5.34 ±4.51 x 105
11 ±9.0
Concrete Placed in PBS and H5N1 virus was spiked
into the tube (not onto the coupon), which
was then agitated on an orbital shaker for
15 min at 200 rpm. The purpose of this
test was to determine if the H5N1 virus was
depositing or absorbing into the concrete
compounded by the highly alkaline extraction
solution.
5.01xl06 3.00 ± 1.74xl05
6.0 ±3.5
Concrete Inoculated with the H5N1 virus and
immediately extracted (placed in PBS,
agitated on an orbital shaker for 15 min at
200 rpm) to prevent the H5N1 virus from
drying out
5.01 x 106 2.44 ±2.38 x 104
0.49 ±0.47
Concrete Placed in sodium phosphate dibasic
heptahydrate + Tween® 80, agitated on an
orbital shaker for 15 min at 200 rpm.
2.00x 106
ND
0.00 ±0.00
Concrete Pulverized immediately after inoculation with
the H5N1 virus, placed in PBS, agitated on
an orbital shaker for 15 min at 200 rpm.
2.00x 106
ND
0.00 ±0.00
Concrete Pulverized immediately after inoculation with
the H5N1 virus, placed in sodium phosphate
dibasic heptahydrate + Tween® 80 agitated
on an orbital shaker for 15 min at 200 rpm.
2.00x 106
ND
0.00 ±0.00
"Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr, except where immediate extraction is noted.
ND = Not detected; the detection limit is 1.31 x 103 TCID_n.
Table 2-6. H5N1 Virus Recovery From Galvanized Metal
Galvanized Metal Placed in PBS, agitated on an orbital
shaker for 15 min at 200 rpm.
7.92x 106
6.96± 1.69x 105
3.8 ±2.1
Galvanized Metal Placed in PBS, agitated on an orbital
shaker for 15 min at 200 rpm.
5.01xl06
12.7 ± 7.42 x 105
25 ± 15
aData are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
-------
Table 2-7. H5N1 Virus Recovery From Glass
Glass Placed in PBS, agitated on an orbital shaker
for 15 min at 200 rpm.
1.98xl06 0.75± l.llx 106 38 ±56
Glass Placed in PBS + 0.01% Tween® 20 (a non-
denaturing surfactant), agitated on an orbital
shaker for 15 min at 200 rpm.
5.01 x 106 1.42 ± 1.52 x 106 28 ± 30
Glass Placed in PBS, agitated on an orbital shaker
for 15 min at 200 rpm.
2.00xl06 1.86± 1.33x 105 9.3 ± 6.7
1 Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
Table 2-8. H5N1 Virus Recovery From Pine Wood
Pine Wood; Placed in PBS, agitated on an orbital shaker
1 cm thickness for 15 min at 200 rpm.
1.98x 106
ND
0.00 ±0.00
Pine Wood; Pine wood soaked in 2% skim milk (dry milk
1 cm thickness reconstituted in deionized water) for 10 min,
allowed to dry to the touch, and autoclaved for
15 min at 121 °C. Once cooled, inoculated
and allowed to dry for 1 hr, then placed in
PBS, agitated on an orbital shaker for 15 min
at 200 rpm.
5.01x 106
ND
0.00 ±0.00
Pine Wood; Placed in PBS, agitated on an orbital shaker
0.2 cm thickness for 15 min at 200 rpm.
2.00x 106
ND
0.00 ±0.00
Pine Wood; Pine wood soaked in 2% skim milk (dry milk
0.2 cm thickness reconstituted in deionized water) for 10 min,
allowed to dry to the touch, and autoclaved for
15 min at 121 °C. Once cooled, inoculated
and allowed to dry for 1 hr, then placed in
PBS, agitated on an orbital shaker for 15 min
at 200 rpm.
2.00x 106
ND
0.00 ±0.00
8 Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
ND = Not detected; the detection limit was 1.31 x 103 TCID_n.
Table 2-9. H5N1 Virus Recovery From Soil
Soil Placed in PBS, agitated on an orbital shaker for
15 min at 200 rpm, then centrifuged with the
supernatant filtered (0.2 urn) for analysis.
1.98x 106
11.0±8.13x 105
56 ±41
Soil Placed in PBS, agitated on an orbital shaker for
15 min at 200 rpm, then centrifuged with the
supernatant filtered (0.2 urn) for analysis.
7.92x 106
4.11 ±2.52x 105
5.2 ±3.2
' Data are expressed as mean + standard deviation.
The drying time or elapsed time between inoculation and recovery was 1 hr.
-------
2.4 Persistence Testing Approach
The TCID50 of the H5N1 vims was measured from the liquid
extracts obtained from test coupons (spiked coupons placed
in the exposure chamber with temperature, RH, UV-A/B
radiation, and contact time treatments) and positive controls
(spiked coupons extracted after the one hour drying time
[time-zero]). Coupon spiking and H5N1 virus extraction
and quantification followed the approach described in
Section 2.3.
The log reduction in TCID50 was calculated as NIN' where
TV is the mean TCID50 from five positive controls of a given
material and N' is the TCID50 from each test coupon replicate
of a given material, environmental condition, and contact
time. The log reduction in TdD50 for each individual test
coupon (R) was calculated for each of the five replicate test
coupons of each material type, environmental condition, and
contact time as:
Equation 2-2.
„.,
*/"*
= log,,
,¥'
Where:
R..,.
ijkl
= log reduction in TdD50 for the /'th replicate
test coupon, y'th test material, Mi environmental
condition, and /th contact time
= arithmetic mean TCID50 from the five positive
controls (which are measured at time-zero) for the
y'th test material
ill-/ = TQD50 recovered on the rth replicate test coupon,
y'th test material, Mi environmental condition, and
/th contact time.
ijkl
If no TCID50 (i.e., no CPE is detected) is measured from a
test coupon (N1), the value one was substituted for N'. Since
the value one is greater than the observed value of zero, the
estimate with this substitution becomes a lower bound for the
true log reduction. Next, the mean log reduction in TCID50
(R) for the five replicate test coupons of a given material/
environmental condition/contact time was calculated as:
Equation 2-3.
Where:
n
= mean log reduction in TCID50 for the j'th test
material, Mi environmental condition, and /th
contact time
= sum of the log reductions in TCID50 for each
individual test coupon for the j'th test material,
Mi environmental condition, and /th contact
time n is the number of test coupon replicates
(five).
The test matrix and various test conditions that were utilized
for H5N1 virus persistence testing are summarized in
Table 2-10. The environmental conditions included various
combinations of temperature, RH, and UV-A/B radiation.
Persistence was measured on four types of test coupons:
chicken feces, galvanized metal, glass, and soil, and the test
durations ranged from one to 13 days. Initial time points
were selected based on comparable data available in the
literature, but subsequent time points were adaptively chosen
(i.e., shorter or longer durations) based on the initial test
results, and tests were not necessarily conducted sequentially
from the shortest to longest test duration.
2,4,1 Temperature
Avian influenza viruses, in general, can survive in the
environment depending on temperature and humidity
conditions, but the various strains of avian influenza may
survive longer in cooler and moister conditions5. The
persistence tests were conducted at room temperature and
a lower temperature to better evaluate the influence of
temperature on the persistence of the H5N1 virus (e.g., the
ability to detect the presence of H5N1 virus). The actual
temperatures associated with each test are provided in
Table 2-10.
The persistence of the H5N1 virus was tested at the room
temperature, low RH, no UV environmental condition by
placing test coupons spiked with the virus into a Plas Labs'
compact glove box under ambient laboratory conditions;
temperature was recorded manually with a digital calibrated
thermometer (Fisherbrand™ Traceable Radio-Signal
Hygrometer/Thermometer, Fisher Scientific, Pittsburgh,
Perm.) at the start and end of the test but not continuously
monitored. The temperature for the other environmental
conditions was recorded continuously at one-minute intervals
with a HOBO® U10 Temperature Data Logger, (Onset
Computer Corporation, Bourne, Mass.).
Low temperature persistence tests (with no UV) were
conducted by placing the coupons inside a sealed
Lock&Lock™ container and the container was then placed
inside a refrigerator. For testing at a low temperature with
UV-A/B, coupons were placed directly beneath the UV-A/B
lamps mounted inside a mini-refrigerator (Marvel® Scientific,
Model No. 6CAR, Greenville, MI) modified to include glove
ports. Some of the temperatures associated with these low
temperature environmental conditions exceeded 20 °C; this
generally occurred during the first two hours of a test as
the temperatures inside the Lock&Lock™ container were
equilibrating (cooling down) relative to the temperature in the
refrigerator. As noted in Section 4.5 and shown in Table 2-10,
the range of temperatures often exceeded the allowable test
measurement tolerance of ± 2 °C (as specified in the test/QA
plan1) for the target temperatures of 22 °C and 4 °C. Although
some of the mean test temperatures deviated from the target
temperatures by a few °C, the associated TCID50 data remain
valid and useful.
-------
Table 2-10. Persistence Test Matrix
Ill JiKiS l!f jil Jffp!
! 1 111
liiliii'iiiliii'ii^^ iifllllllllllllllllllllllllM
! Ill 1
)
Room Temperature (22 °C), Low
RH (40%), No UVa
1-Day
2- Day
4- Day
Start
Chicken feces, galvanized metal, 22 7
glass, and soil
22.0
23.2
End Start
23.4 32
22.9 30
22.8 36
End
38
42
61
Room Temperature (22 °C), High
RH (80%), No UV
1-Day
2-Day
4- Day
Mean
Range Mean
Chicken feces, galvanized metal, 224 190-232 891
glass, and soil
23.4 22.6-24.4 90.4
23.7 22.7-24.9 91.2
Range
51.9-93.5
80.3-93.8
76.5-94.4
Low Temperature (4 °C),Low RH
(40%), No UV
4- Day
8- Day
13-Day
Low Temperature (4 °C), High
(80%), No UV
4- Day
9- Day
13-Day
Mean
Chicken feces, galvanized metal, 4 Q 3
glass, and soil
6.7 6
6.7 6
RH
Mean
Chicken feces, galvanized metal, 7 g 7
glass, and soil
6.7 6
6.8 6
Range Mean
41-21.9 27.7
46-22.2 15.2
36-22.6 46.3
Range Mean
22-23.1 89.5
38-22.9 96.9
48-22.6 79.0
Range
17.5-33.3
1.00-71.3
27.4-54.6
Range
63.6-92.8
66.4- 104
24.9-100
Low Temperature (4 °C), Low RH
(40%), UV-A/B
24-Hours
48-Hours
96-Hoursb
Mean
Chicken feces, galvanized metal, 4 p 2
glass, and soil
Range Mean
26-20.8c 24.5b
-0.91 -2.04-21.2 29.7
0.12 -1
.76-21.7 28.2
Range
23.1-68.3C
28.0-86.2
25.2-65.2
" For the room temperature, low RH, no UV test condition, temperature was recorded manually and RH was measured with a digital calibrated
hygrometer. Both temperature and RH were recorded at the start and end of the test; neither was continuously monitored.
b Galvanized metal and glass were not tested at the 4-day test duration.
c Temperature and RH were not recorded during the initial seven hours of the test.
2.4.2 Relative Humidity
Persistence testing was conducted using low and high
RH levels to better understand the influence of RH on
the persistence of the H5N1 virus. The actual RH levels
associated with each test are provided in Table 2-10; the
range of RH levels often exceeded the allowable test
measurement tolerance of ±10% specified in the test/QA
plan1, as noted in Section 4.5. Deviations from the target RH
levels do not invalidate the associated TdD50 data.
For the room temperature, low RH, no UV environmental
condition, persistence of the H5N1 virus was tested at
ambient laboratory conditions (no active measures were
taken to increase or reduce the RH) inside a glove box; RH
was measured at the start and end of the test with a digital
calibrated hygrometer but not continuously monitored.
RH for the persistence testing performed under other
environmental conditions was recorded continuously at
one-minute intervals with a device that measures and logs
temperature and RH data (HOBO® U10, Onset Computer
Corporation). For the room temperature, low RH, no UV
environmental condition, the RH level appeared to increase
with the duration of the test. The moisture content associated
with the materials being tested (e.g., soil and chicken feces)
likely contributed to the increasing RH inside the sealed
glove box as time elapsed.
Persistence testing at the room temperature, high RH, no UV
environmental condition was accomplished by placing the
coupons directly in a pre-humidified (by using an ultrasonic
fogger) glove box. The RH tended to increase with the
placement of the coupons into the glove box; consequently
during the 1-day test, fresh air was introduced into the glove
box to manually lower the RH at the start of the 1-day test.
During the 2- and 4-day tests, there were no attempts to
adjust the RH, which increased during testing.
Persistence testing at low temperature (with no UV)
was conducted by placing the coupons inside a sealed
Lock&Lock™ container inside a refrigerator. When testing
at a low RH, DRIERITE (W. A. Hammond DRIERITE
Co., Xenia, Ohio) was also added to the container. In an
attempt to prevent the RH from increasing as the DRIERITE
became saturated, fresh DRIERITE was exchanged with
the used DRIERITE on the fourth day of the 8-day test
and this reduced the RH from 20-30% to <10% (the RH
briefly spiked to 71.30% during the exchange). For high RH
-------
persistence testing (target RH 80% per the test/QAplan1)
inside the Lock&Lock™ container at low temperature,
mean RH levels ranging from 79% to 97% were achieved by
sealing the inherently moist materials (i.e., soil and chicken
feces) in the container.
For testing with UV-A/B (low temperature, low RH), the RH
inside the modified mini-refrigerator remained relatively low
throughout the testing such that the use of DRIERITE was
not required.
2.4.3 Ultraviolet-A/B Radiation
Influenza viruses may be inactivated by UV-B wavelengths
in sunlight6. UV-A/B radiation was implemented in the tests
in an effort to mimic UV radiation levels associated with
natural sunlight. The spectrum and intensity of terrestrial
UV radiation is highly variable and is affected by time of
day, day of year, geographical location, altitude, atmospheric
pollution, and clouds. Naturally occurring UV-B levels,
observed around noon, range from 19.5 to 150 microwatt
(uW)/cm2,7~10, and therefore a target exposure chamber
condition of 70 uW/cm2 UV-B was selected. The amount of
UV-A generated during testing was quantitated during the
method development phase and actual testing and was kept
within the range of UV-A observed in natural sunlight (0 to
4,500 uW/cm2)11. The target UV-C level to be generated
during testing was 0 uW/cm2, since UV-C generally does
not reach the earth's surface. UV-B, UV-C, and total UV
were monitored during the persistence testing (UV-A was
calculated as total UV minus the UV-B and UV-C levels).
The level of UV-B radiation generated and the potential
of diminishing UV-B output over time was evaluated
with data obtained from the lamp manufacturer and data
generated during the method development phase of testing.
AReptiSun™ 10.0 Linear Fluorescent UV-B Lamp
was used in the study. The manufacturer, Zoo
Med Laboratories, Inc. (San Luis Obispo, Calif.)
indicated that after an initial burn in, UV-B levels of
the lamp would diminish somewhat over time.
Using a single ReptiSun™ 10.0 Linear Fluorescent
UV-B Lamp with a baseline UV-B level of 70 uW/cm2,
and a Solarmeter® Model 6.2 (Solartech, Inc., Harrison
Township, Mich.) the following UV-B levels were measured
sequentially overtime during preliminary tests:
• 69 uW/cm2, 99% of baseline remaining after 1 hour
• 68 uW/cm2, 97% of baseline remaining after 2 hours
• 66 uW/cm2, 94% of baseline remaining after 17 hours
• 64 uW/cm2, 91% of baseline remaining after 64 hours
• 64 uW/cm2, 91% of baseline remaining after 89 hours
The UV-A/B lamps were placed inside a scientific-grade
mini-refrigerator, which was customized with a faceplate and
glove ports (see Figure 2-3). All UV-A/B exposures were
continuous; the lamps were not turned off during testing.
In addition to measuring UV-B, UV-C was measured with
the Solarmeter® Model 8.0 and total UV was measured
with Solarmeter® Model 5.0. UV data measured during the
persistence testing are summarized in Table 2-11. During
the 24-hour test, UV measurements were recorded from the
center of the testing area at the start and completion of the
test. During the 48- and 96-hour tests, UV measurements
were periodically made from five positions beneath the lamps
(Figure 2-4). UV measurements were taken at approximately
14 cm below the lamps, which corresponded to the distance
of the test coupons from the lamps. All UV-B levels
coincided with the target UV level (70 ± 7 uW/cm2 UV-B as
specified in the test/QA plan).
Figure 2-3. Mini-Refrigerator Configured With Ultraviolet A/B Lamps
-------
Table 2-11. Ultraviolet Measurements
24-Hour1
48-Hourc
Mean
109
Range
100-122
Mean
68
Range
63-74
Mean
0
Range
0-0
96-Hourc
Mean
106
Range
97- 120
Mean
69
Range
63-75
Mean
0
Range
0-0
UV-A/B exposures for the 24-, 48-, and 96-hour tests were continuous.
' During the 24-hour test, UV measurements were recorded from the center of the testing area at the start and completion of the test.
During the 48- and 96-hour tests, UV measurements were periodically made from five positions beneath the lamps (see Figure 2-4).
Mini-fridge work
surface (top view)
Five points measured for UV-B, UV-C and
total UV intensity during persistence trial
All coupons located within dashed line
Figure 2-4. Schematic (Top View) of Ultraviolet Sampling Locations
2.5 Test Results
Persistence results for each material/environmental condition
combination are summarized in Tables 2-12 through 2-15.
A summary of all persistence data obtained is provided in
Table 2-16.
2.5.1 Chicken Feces
The results obtained for persistence of the H5N1 virus on
chicken feces are summarized in Table 2-12.
The H5N1 virus was least persistent (6.3 mean log reduction
in TCID50 during the 2-day test) on chicken feces for the
room temperature, low RH, no UV environmental condition,
and the H5N1 virus was not detected above the TCID50 on
the laboratory and procedural blanks at any test duration.
Extracts from chicken feces samples not inoculated with
the H5N1 virus often induced CPE when testing the
most concentrated serial dilution (no CPE observed with
subsequent dilutions). This CPE often resulted in a TdD50
of 3.16 x 103, and was attributed to the inherent properties of
the chicken feces rather than the H5N1 virus. Test coupons
with mean recovered TdD50 values less than or equal to the
associated laboratory and procedural blanks were still used in
the calculation of the mean log reductions.
At higher RH (room temperature, high RH, no UV
environmental condition), the mean TCID50 level recovered
from the test coupons after the 1-day test was greater than the
TdD50 levels associated with the laboratory and procedural
blanks. Mold was observed growing on the chicken feces
during the 4-day test at the room temperature, high RH, no
UV environmental condition.
-------
Table 2-12. H5N1 Virus Persistence on Chicken Feces
IlllllSSfi**!^
1-Day
Positive Control"
Test Coupon6
Laboratory Blank*
Procedural Blank6
2-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
ii in
.*:di
Room
7.94x 106
7.94x 106
0
0
7. 94 xlO6
7.94x 106
0
0
5.01 x 106
5. 01 xlO6
0
0
i 11 11 ,.i is^-^^iiiiS^nl^^^^K.-T'iSifp^f f
}h i-liii
Temperature, Low RH, No UV
5/5 8. 05 ±4. 16 x 105 10.1 ±5. 2
5/5 6. 37 ±3. 99 x 102 h 0.01 ± 0.01h 3.2 ± 0.26h
1/1 3. 16 xlO3
1/1 3.16xl03
5/5 2.11 ± 1.95 xlO6 26.6 ±24.5
0/5 ND1 0.00±0.00i 6.3±0.00i
1/1 3. 16 xlO3
1/1 3.16xl03
5/5 3.66± 1.87x 105 7. 30 ±3. 73
0/5 ND1 0.00±0.00i 5.6±0.00i
1/1 3.16xl03
1/1 3. 16 xlO3
Room Temperature, High RH, No UV
1-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
4-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5. 01 xlO7
5. 01 xlO7
0
0
3.16x 107
3. 16 xlO7
0
0
2. 00 xlO7
2.00x 107
0
0
5/5 6.96 ±3. 96 xlO6 13.9 ± 7.90
5/5 7. 70 ±3. 11 xlO4 0.15 ±0.06 2.0 ± 0.2
1/1 3.16xl03
1/1 3. 16 xlO3
5/5 2.54± 1.59x 105 0.80 ± 0.50
5/5 3.16±0.00xl03h 0.01±0.00h 1.9±0.0h
1/1 3.16xl03
1/1 3. 16 xlO3
5/5 1.39 ± 1.17 xlO6 6. 96 ±5.83
0/5 ND1 0.00±0.00i 6.1±0.0i
1/1 3. 16 xlO3
1/1 3.16xl03
Low Temperature, Low RH, No UV
4-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
8-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
13-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00x 107
2.00x 107
0
0
5. 01 xlO7
5.01 x 107
0
0
2.00x 107
2. 00 xlO7
0
0
5/5 2.02± 1.83x 106 10.1 ±9. 13
5/5 7.11 ±3.32x 104 0.36±0.17 1.5 ±0.2
1/1 3. 16 xlO3
1/1 3.16xl03
5/5 8.05 ±4. 16 xlO5 1.61 ±0.83
5/5 3.16±0.00x 103h 0.01±0.00h 2.4 ± 0.0h
1/1 3. 16 xlO3
1/1 3.16xl03
5/5 7. 10 ±3. 94 x 105 3. 55 ±1.97
0/5 ND1 0.00±0.00i 5.9±0.0i
1/1 3.16xl03
0/1 ND
-------
Table 2-12. H5N1 Virus Persistence on Chicken Feces (continued)
S8S8S8S8s«rf^^
11131= Fji II!|||||||I|||||||I||"?W. «3*"SPW*»*;«*S^
Low Temperature, High RH, No UV
4-Day
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
9-Day
Positive Control 7.94 x 106
Test Coupon 7.94 x 106
Laboratory Blank 0
Procedural Blank 0
5/5 8. 05 ±4. 16 x 105 1.61 ±0.83
5/5 5.42 ± 4.09 x 104 0.11 ±0.08 1.3 ±0.3
1/1 3.16xl03
1/1 3.16xl03
5/5 4.42 ± 4.86 x 105 5.57 ±6. 12
5/5 5. 90 ±4. 57 x 104 0.74 ± 0.58 1.0 ±.05
1/1 3.16xl03
1/1 3.16xl03
13-Day
Positive Control 2.00xl07 5/5 7.10 ± 3.94 x 105 3. 55 ±1.97
Test Coupon 2.00xl07 3/5 7.86 ± 7.17 x 102 0.00 ± O.OO1 4.0 ± 2. 01
Laboratory Blank 0 1/1 3.16xl03
Procedural Blank 0 0/1 ND
Low Temperature, Low RH, UV-A/B
24-HouH
Positive Control 2.00 x 107
Test Coupon 2.00 x 107
Laboratory Blank 0
Procedural Blank 0
48-Hour'
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
96-Hour'
Positive Control 2.00 x 107
Test Coupon 2.00 x 107
Laboratory Blank 0
Procedural Blank 0
5/5 7. 10 ±3. 94 x 105 3. 55 ±1.47
5/5 1.06±0.86x 106 5.32 ± 4.29 -0.05 ± 0.37
1/1 3.16xl03
1/1 3.16xl03
5/5 1.07 ±0.26 x 106 2. 14 ±0.51
5/5 9. 58 ± 4.16 x 104 0.19 ±0.08 1.1 ±0.22
1/1 3.16xl03
1/1 3.16xl03
5/5 2.07 ± 1.08 x 106 10.4 ± 5.40
5/5 3.16±0.00x 103h 0.02±0.00h 2.8 ± 0.0h
1/1 3.16xl03
1/1 3.16xl03
"CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c TCID50 values for laboratory and procedural blanks are attributed to CPE observed from the test material rather than H5N1 virus.
d Positive controls were inoculated, extracted at time-zero (1-hour drying time).
e Test coupons were inoculated, exposed to the environmental condition for the test duration.
f Laboratory blanks were not inoculated with any virus, and extracted at time-zero.
B Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.
h The test coupon TCID50 was < the procedural blank TCID50 such that the CPE observed may be attributed to the test material rather than
H5N1 virus.
A value of 1 TCID50 was used for each test coupon replicate with no CPE observed, in the calculation of % virus recovery and mean log reduction.
J UV-A/B exposures for 24-, 48-, and 96-hour tests were continuous.
ND = Not detected; the detection limit was 1.31 x 103 TCID50.
"-" Not applicable.
Lowering the temperature resulted in longer persistence times
(1.5 mean log reduction in the TCID50 during the 4-day test).
In fact, the H5N1 virus was recovered (detected above the
laboratory and procedural blank level) from chicken feces
after the 4-day test at the low temperature, low RH, no UV
environmental condition and after the 9-day test at the low
temperature, high RH, no UV environmental condition.
Even with exposure to UV-A/B (at a low temperature and
low RH), the H5N1 virus was detected above the laboratory
and procedural blank level after 48 hours of continuous UV-
A/B exposure test on chicken feces.
-------
2.5.2 Galvanized Metal
The test results obtained for persistence of the H5N1 virus
on galvanized metal are summarized in Table 2-13. The
H5N1 virus was not detected in any of the tests conducted
at room temperature. When the tests were conducted at the
low temperature, low RH, no UV environmental condition,
the H5N1 virus was detected after the 13-day test with only a
0.69 mean log reduction in TCID50. At the low temperature,
high RH, no UV environmental condition, the H5N1 virus
was only detected after a 4-day test; during the 9-day test,
the galvanized metal appeared to exhibit oxidation (black
spots formed where the H5N1 virus was inoculated), but the
H5N1 virus was not detected. When testing under high RH
conditions, it was common for the dried inoculum drops to
reform (rehydrate) into liquid drops on non-porous surfaces
(e.g., galvanized metal). When exposed to UV-A/B (at a low
temperature and low RH), the H5N1 virus persisted only for
the 24-hour test.
Table 2-13. H5N1 Virus Persistence on Galvanized Metal
«H",i^J'*WW*W',^''i»i»i"'''l:ll (ill Bill 1 1
ilili! ihW:i.&^:U?.!:MU!.V*i- ' '! i! '""• ^^^ i'lili: iii^j : iiii Igiiii I: :
VfSffSffSff&tkiW^ih'i!^^
i 81, s i isisisiK
^itisy^i ;M;il!"x i 3iiS^
Room Temperature, Low RH, No UV
1-Day
Positive Control0 7.94xl06
Test Coupond 7.94xl06
Laboratory Blank6 0
Procedural Blank* 0
2-Day
Positive Control 7.94xl06
Test Coupon 7.94xl06
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 5.01 x 106
Test Coupon 5.01 x 106
Laboratory Blank 0
Procedural Blank 0
5/5 1.56±0.41x 106 19.6 ±5.10
0/5 ND6 0.00 ± O.OQs 6.2 ± O.Q6
0/1 ND
0/1 ND
5/5 2.25± 1.28x 106 28.3 ± 16.1
0/5 ND6 0.00 ±0.008 6.4 ±0.06
0/1 ND
0/1 ND
5/5 1.36± l.OSx 106 27. 2 ±20. 5
0/5 ND6 0.00 ±0.006 6.1 ±0.08
0/1 ND
0/1 ND
Room Temperature, High RH, No UV
1-Day
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
2-Day
Positive Control 3.16xl07
Test Coupon 3.16xl07
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
5/5 6. 16 ±3. 69 x 106 12.3 ± 7.36
0/5 ND6 0.00 ±0.006 6.8 ±0.06
0/1 ND
0/1 ND
5/5 1.13 ±0.63 x 106 3. 56 ±1.98
0/5 ND6 0.00 ±0.006 6.1 ±0.06
0/1 ND
0/1 ND
5/5 2. 76 ±2. 05 x 106 13.8 ± 10.3
0/5 ND6 0.00 ± 0.006 6.4 ± O.Q6
0/1 ND
0/1 ND
-------
Table 2-13. H5N1 Virus Persistence on Galvanized Metal (continued)
4-Day
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
8-Day
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
13-Day
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
9-Day
Positive Control 7.94xl06
Test Coupon 7.94xl06
Laboratory Blank 0
Procedural Blank 0
13-Day
Positive Control 2.00 x 107
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
24-Hoursh
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
48-Hours"
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
fat WJ-H;I 1 l||f 1 If ;>ff 1 1 1
;,ii^A\Ai*&\»"ms^ n n n
Low Temperature, Low RH, No UV
5/5 2.92± 1.42x 106 14. 6 ±7. 11
5/5 3. 78 ±2. 80 x 105 1.89 ±1.40 1.0 ±0.38
0/1 ND
0/1 ND
5/5 2.08±0.68xl06 4.16 ±1.36
5/5 7. 33 ±3. 58 x 105 1.46 ±0.71 0.50 ± 0.23
0/1 ND
0/1 ND
5/5 1.56 ±0.41 x 106 7. 78 ±2. 03
5/5 3.44 ± 1.51x10= 1.72 ±0.76 0.69 ± 0.20
0/1 ND
0/1 ND
Low Temperature, High RH, No UV
5/5 2.08±0.68xl06 4.16 ±1.36
5/5 4.69±3.06xl03 0.01 ± 0.01 2.7 ± 0.3
0/1 ND
0/1 ND
5/5 8.81 ±6.58 x 105 11.1 ±8. 28
0/5 NDB 0.00 ±0.006 5.9 ± O.OB
0/1 ND
0/1 ND
5/5 1.56 ±0.41 x 106 7. 78 ±2. 03
0/5 NDB 0.00 ± 0.008 6.2 ± 0.0^
0/1 ND
0/1 ND
Low Temperature, Low RH, UV-A/B
5/5 1.56 ±0.41 x 106 7. 78 ±2. 03
5/5 0.64 ± 1.41 xlO5 0.32 ±0.70 2.6 ±1.1
0/1 ND
0/1 ND
5/5 1.56±0.41xl06 3. 10 ±0.81
0/5 NDs 0.00 ± O.OQs 6.2 ± 0.0^
0/1 ND
0/1 ND
a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls were inoculated, extracted at time-zero (1-hour drying time).
d Test coupons were inoculated, exposed to the environmental condition for the test duration.
e Laboratory blanks were not inoculated with any virus, and extracted at time-zero.
f Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.
8 A value of 1 TCIDso was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction.
h UV-A/B exposures for the 24- and 48-hour tests were continuous.
ND = Not detected; the detection limit was 1.31 x 103 TCID50.
"-" Not applicable.
-------
2.5.3 Glass
The H5N1 virus persistence results on glass are summarized
in Table 2-14. The H5N1 virus was not detected on any of
the coupons tested at room temperature. When the tests were
conducted at the low temperature, low RH, no UV environmental
condition, the H5N1 virus was detected after a 13-day test with
only a 0.59 mean log reduction in TCID50. When the tests were
conducted at the low temperature, high RH, no UV environmental
condition, the H5N1 virus was detected on glass after the 9-day
test with a 2.7 mean log reduction in TCID50. As noted previously
for galvanized metal, when testing under high RH conditions, it
was common for the dried inoculums to reform (rehydrate) into
liquid drops on non-porous surfaces (e.g., glass). When exposed
to UV-A/B (at a low temperature and low RH), the H5N1 virus was
detected on only one out of five replicate coupons following the
24-hour test.
Table 2-14. H5N1 Virus Persistence on Glass
!SiiSi-?W'Hti[f*!iiiiiiiiiif'f;''f y ffftvh
«i»»tete«Ai,!,u3SSSSSS^^ :,;;«:,;,X,;<
1-Day
Positive Control0 7.94x
Test Coupond 7.94x
Laboratory Blank6 0
Procedural Blank' 0
2-Day
Positive Control 7.94x
Test Coupon 7.94x
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 5.01 x
Test Coupon 5.01 x
Laboratory Blank 0
Procedural Blank 0
1-Day
Positive Control 5.01 x
Test Coupon 5.01 x
Laboratory Blank 0
Procedural Blank 0
2-Day
Positive Control 3.16x
Test Coupon 3.16x
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 2.00x
Test Coupon 2.00x
Laboratory Blank 0
Procedural Blank 0
>4>il«toM3S;&i5Si\*,A^ K K K MbihoiutnAilaiaJi/St
Room Temperature, Low RH, No UV
106 5/5 6.18 ± 1.60x10= 7.79 ± 2.02
106 0/5 ND6 0.00 ± 0.006 5.3 ± O.Q6
0/1 ND
0/1 ND
106 5/5 3.29± 1.71 x 106 41. 4 ±21. 6
106 0/5 ND6 0.00 ±0.006 6.5 ± O.Q6
0/1 ND
0/1 ND
106 5/5 1.01 ±0.63 xlO6 20. 2 ±12. 7
106 0/5 ND6 0.00 ± O.OQs 6.0 ± O.Q6
0/1 ND
0/1 ND
Room Temperature, High RH, No UV
107 5/5 1.07±0.26x 107 21. 4 ±5. 09
107 0/5 ND6 0.00 ±0.006 7.0 ± O.Q6
0/1 ND
0/1 ND
107 5/5 2.66±2.21xl06 8.43 ± 7.01
107 0/5 ND6 0.00 ± 0.006 6.4 ± O.Q6
0/1 ND
0/1 ND
107 5/5 2.32±0.83x 106 11. 6 ±4. 14
107 0/5 ND6 0.00 ±0.006 6.4 ± O.Q6
0/1 ND
0/1 ND
llrf.ll
-------
Table 2-14. H5N1 Virus Persistence on Glass (continued)
Low Temperature, Low RH, No UV
4-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00x 107
2.00x 107
0
0
5/5
5/5
0/1
0/1
1.07 ±0.59x 106
4.98 ±2.97 x 105
ND
ND
5.35 ±2.93
2.49 ± 1.49
0.41 ±0.30
8-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
5/5
0/1
0/1
1.10±0.63x 106
1.34 ±2.07 x 106
ND
ND
2.20 ± 1.25
2.68 ±4.12
0.26 ±0.58
13-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00 x 107
2.00 x 107
0
0
5/5
5/5
0/1
0/1
1.79±0.83x 106
5.91 ±4.48x 105
ND
ND
8.94 ±4.16
2.96 ±2.24
0.59 ±0.36
Low Temperature, High RH, No UV
4-Day
Positive Control
9-Day
Positive Control
13-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
7.94 x 105
2.00 x 107
2.00 x 107
0
0
5/5
1.34± 1.09x 106
2.67 ±2.17
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
0
0
5/5
0/1
0/1
1.37 ± 0.61 x 105 0.27 ±0.12
ND
ND
0.63 ±0.37
-
-
5/5
1.58± 1.09 x 105
19.9 ± 13.8
Test Coupon
Laboratory Blank
Procedural Blank
7.94x 106
0
0
5/5
0/1
0/1
3.16±0.00x 103 0.04 ±0.00
ND
ND
2.7 ±0.0
-
-
5/5
0/5
0/1
0/1
1.79±0.83x 106
ND6
ND
ND
8.94 ±4.16
0.00 ±0.008
6.3 ±0.06
Low Temperature, Low RH, UV-A/B
24-Hour"
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00 x 107
2.00x 107
0
0
5/5
1/5
0/1
0/1
1.79±0.83x 106
2.62 ±5.85 x 1026
ND
ND
1.83 ±0.66
0.00 ±0.006
5.6 ± 1.46
48-Hourh
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
0/5
0/1
0/1
1.31 ±0.43x 106
ND6
ND
ND
2.62 ±0.86
0.00 ±0.006
6.1 ±0.06
"CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls were inoculated, extracted at time-zero (1-hour drying time).
d Test coupons were inoculated, exposed to the environmental condition for the test duration.
e Laboratory blanks were not inoculated with any virus, and extracted at time-zero.
f Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.
B A value of 1 TCID50 was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction.
h UV-A/B exposures for the 24- and 48-hour tests were continuous.
ND = Not detected; the detection limit was 1.31 x 103 TCID50.
"-" Not applicable.
-------
2.5.4 So/7
The H5N1 vims persistence results in soil are summarized
in Table 2-15. When the tests were conducted at room
temperature, the H5N1 virus was detected after the
1-day test, but not at the longer durations. When the tests
were conducted at the low temperature, low RH, no UV
environmental condition, the H5N1 virus was detected
from soil after a 13-day test with a 3.1 mean log reduction
Table 2-15. H5N1 Virus Persistence on Soil
in TCID50. When the tests were conducted at the low
temperature, high RH, no UV environmental condition, the
H5N1 virus was detected from soil after the 9-day test with
no mean log reduction in TCID50. When exposed to UV-A/B
(at a low temperature and low RH), the H5N1 virus persisted
during the 96-hour test with a 3.3 mean log reduction in
TCID50.
iSala.
1-Day
Positive Control" 7.94xl06
Test Coupon6 7.94xl06
Laboratory Blank* 0
Procedural Blank6 0
2-Day
Positive Control 7.94xl06
Test Coupon 7.94xl06
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 5.01 x 106
Test Coupon 5.01 x 106
Laboratory Blank 0
Procedural Blank 0
1-Day
Positive Control 5.01 x 107
Test Coupon 5.01 x 107
Laboratory Blank 0
Procedural Blank 0
2-Day
Positive Control 3.16xl07
Test Coupon 3.16xl07
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
4-Day
Positive Control 2.00xl07
Test Coupon 2.00xl07
Laboratory Blank 0
Procedural Blank 0
.'iwSliSa'&j^^fcattm li lili MsfesiSfssslftjlfsriifJ
Room Temperature, Low RH, No UV
5/5 7.33 ±3.58x10= 9. 23 ±4. 51
2/5 0.88± 1.38x 104h 0.11±0.17h 4.2 ± 2.4h
0/1 ND
0/1 ND
5/5 6. 40 ±2. 21 x 105 8.06 ± 2.78
0/5 NDh 0.00±0.00h 5.8±0.0h
0/1 ND
0/1 ND
5/5 2.55±0.88x 105 5. 09 ±1.75
0/5 NDh 0.00±0.00h 5.4±0.0h
0/1 ND
0/1 ND
Room Temperature, High RH, No UV
5/5 1.48 ± 1.12 xlO6 2. 96 ±2. 24
5/5 8.26 ± 4.31 x 104 0.16 ±0.09 1.3 ±0.26
0/1 ND
0/1 ND
5/5 3. 64 ± 2.80xl05 1.15 ±0.89
0/5 NDf 0.00 ± 0.00f 5.6 ± 0.0f
0/1 ND
0/1 ND
5/5 1.79 ±0.83x10= 0.89 ± 0.42
0/5 NDf 0.00 ± 0.00f 5.3 ± 0.0f
0/1 ND
0/1 ND
Low Temperature, Low RH, No UV
5/5 2.82 ± 1.57x10= 1.41 ±0.78
5/5 2. 47 ±3. 29 x 104 0.12 ±0.16 1.5 ±0.73
0/1 ND
0/1 ND
-------
Table 2-15. H5N1 Virus Persistence on Soil (continued)
Low Temperature, Low RH, No UV
8-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
5/5
0/1
0/1
5.23 ± 1.72x 105
1.16±0.14x 103
ND
ND
1.04 ±0.34
0.00 ±0.00
2.7 ±0.1
13-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00x 107
2.00 x 107
0
0
5/5
4/5
0/1
0/1
3.67 ± 1.31 x 105
1.05 ± 0.59 x 103h
ND
ND
1.83 ±0.66
0.01±0.00h 3.1±1.4h
-
-
Low Temperature, High RH, No UV
4-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
5/5
0/1
0/1
5.23± 1.72x 105
2.21 ± 1.65x 105
ND
ND
1.04 ±0.34
0.44 ± 0.33
0.46 ±0.30
9-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
7.94x 106
7.94 x 106
0
0
5/5
5/5
0/1
1/1
2.82 ±3.02x 105
5. 93 ±3. 95 x 105
ND
2.00xl04
3. 56 ±3. 81
7.46 ±4.98
-
-
-0.25 ±0.28
-
-
13-Day
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00 x 107
2.00x 107
0
0
5/5
0/5
0/1
0/1
3.67± 1.31 x 105
NDh
ND
ND
1.83 ±0.66
0.00±0.00h
5.6±0.0h
Low Temperature, Low RH, UV-A/B
24-Hours1
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00x 107
2.00 x 107
0
0
5/5
5/5
0/1
0/1
3.67 ± 1.31 x 105
5.93 ±3.95 x 105
ND
ND
3.55± 1.97
2.96 ± 1.98
-0.14 ±0.28
48-Hours1
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
5/5
0/1
0/1
5.23± 1.72x 105
3.90± l.Olx 104
ND
ND
1.04 ±0.34
0.08 ±0.02
1.1 ±0.1
96-Hours1
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00x 107
2.00 x 107
0
0
5/5
4/5
0/1
0/1
1.07 ±0.59x 106
3.11 ±3.28x 103h
ND
ND
5.35 ±2.93
0.02±0.02h
3.3 ± 1.6h
a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c TCID50 values for laboratory and procedural blanks are attributed to CPE observed from the test material rather than H5N1 virus.
d Positive controls were inoculated, extracted at time-zero (1-hour drying time).
e Test coupons were inoculated, exposed to the environmental condition for the test duration.
f Laboratory blanks were not inoculated with any virus, and extracted at time-zero.
8 Procedural blanks were not inoculated, but exposed to the environmental condition for the test duration.
h A value of 1 TCIDso was used when no CPE are observed, in the calculation of % virus recovery and mean log reduction.
UV-A/B exposures for the 24-, 48-, and 96-hour test were continuous.
ND = Not detected; the detection limit was 1.31 x 103 TCID50.
"-" Not applicable.
-------
2.5.5 Comparison of Chicken Feces, Galvanized Metal,
Glass, and Soil Results
The H5N1 vims persistence results are summarized in
Table 2-16. These data denote 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 H5N1
virus remained viable for each material and environmental
condition. The H5N1 virus persisted for relatively short
durations on all materials at room temperature, low RH, no
UV and at room temperature, high RH, no UV. The H5N1
virus was not detected after the 1-day test on galvanized
metal and glass and was only detected after the 1-day test on
soil. For chicken feces, at the room temperature, high RH,
no UV environmental condition, the TdD50 level measured
after the 2-day test was less than the TCID50 associated with
the procedural blank, possibly indicating an effect of the test
material, rather than the H5N1 virus. Similarly, for the room
temperature, low RH, no UV environmental condition, the
TCID50 level measured from chicken feces after the 1-day
test was less than the associated procedural blank TCID50,
indicating a possible test material interference rather than the
H5N1 virus.
The H5N1 virus persisted longer on all materials at both
low temperature conditions (i.e., low temperature, low RH,
no UV and low temperature, high RH, no UV) compared to
the room temperature conditions. For the low temperature,
low RH, no UV environmental condition, the H5N1 virus
persisted on galvanized metal, glass, and soil for 13 days (the
longest duration tested). At the low temperature, high RH, no
UV environmental condition, the H5N1 virus persisted for
shorter durations on galvanized metal (four days), glass (nine
days), and soil (nine days). Interestingly, the H5N1 virus
persisted on chicken feces longer at the low temperature, high
RH, no UV environmental condition (13 days) than at the
low temperature, low RH, no UV environmental condition
(possibly eight days where the TCID50 level detected was
less than the TCID50 associated with the procedural blank,
possibly indicating an effect of the test material).
Table 2-16. Summary of H5N1 Virus Persistence
Chicken feces
III JJP
Room temperature, low RH, no UV la 2
Room temperature, high RH, no UV 2a 4
Low temperature, low RH, no UV 8a 13
Low temperature, high RH, no UV 13 NA
Low temperature, low RH, UV-A/B 4a NA
Galvanized metal
Room temperature, low RH, no UV NA 1
Room temperature, high RH, no UV NA 1
Low temperature, low RH, no UV 13 NA
Low temperature, high RH, no UV 4 9
Low temperature, low RH, UV-A/B 1 2
Glass
Room temperature, low RH, no UV NA 1
Room temperature, high RH, no UV NA 1
Low temperature, low RH, no UV 13 NA
Low temperature, high RH, no UV 9 13
Low temperature, low RH, UV-A/B 1 2
Soil
Room temperature, low RH, no UV 1 2
Room temperature, high RH, no UV 1 2
Low temperature, low RH, no UV 13 NA
Low temperature, high RH, no UV 9 13
Low temperature, low RH, UV-A/B 4 NA
a The TCIDso measured was at a level < the procedural blank TCIDso for chicken feces such that the CPEs observed (and used in the calculation
of the TCID50) may be attributed to the test material rather than H5N1 virus.
NA = Not available; the H5N1 virus was either detected at all durations or not detected from any duration.
-------
For the low temperature, low RH, UV-A/B environmental
condition, the H5N1 virus persisted longer on chicken feces
and soil than galvanized metal and glass. The H5N1 virus
was detected after only one day (24 hours of continuous
exposure to UV-A/B) from galvanized metal and glass.
The H5N1 virus was detected after two to four days of
continuous exposure to UV-A/B on chicken feces and soil;
on chicken feces the H5N1 virus was detected after the 2-day
test at a level above the associated procedural blank and
potentially detected after the 4-day test but at a level below
the associated procedural blank, possibly indicating an effect
of the test material rather than the H5N1 virus. In comparing
these results to the low temperature, low RH environmental
condition (no simulated sunlight), the UV-A/B clearly
diminishes the H5N1 persistence.
-------
-------
3.0
Decontamination Technology Evaluation
3.1 Technology Descriptions
The liquid decontamination technologies evaluated consisted
of:
• 1% Citric Acid
o 1% citric acid was prepared by adding 1 gram (g)
citric acid, anhydrous, (> 99.5% purity) to 99 mL
hard water until completely dissolved.
• pH-Amended Bleach
o pH-amended bleach was prepared by adding 5%
acetic acid to household bleach (Clorox®; 5-6%
sodium hypochlorite) to obtain a pH-amended bleach
solution. The solution was prepared using 9.4 parts
hard water, 1 part bleach, and 1 part 5% glacial acetic
acid to obtain a solution having a mean pH of 6.81
± 0.15 and a mean total chlorine content of 6,215 ±
212ppm.
• 732 ppm Quaternary Ammonium
o 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.
•8%Na2CO3
o 8% Na2CO3 was prepared by adding 8 g Na2CO3
(SigmaUltra (Sigma-Aldrich Inc.,St. Louis, Mo.);
>99.0% purity) to 92 mL hard water until completely
dissolved.
All preparations and dilutions were made using AOAC
International hard water prepared at 400 ppm hardness as
calcium carbonate (CaCO3) (AOAC Official Method 960.09,
Germicidal and Detergent Sanitizing Action of Disinfectants,
Section E. Synthetic Hard-Water, p. 11). All decontamination
contact times were 10 minutes.
3.2 Cytotoxicity of Neutralized Decontamination
Liquids
During the decontamination technology evaluation,
neutralization of each decontamination liquid was required
in order to terminate activity at the end of the 10-minute
decontamination contact time. For neutralization to be
acceptable, the chemicals used could not interfere with the
methods used for extraction and quantification of the H5N1
virus. The potential for neutralized decontamination liquids
to cause cytotoxicity to the MDCK cells, which could
subsequently interfere with the TCID50 determination, was
assessed with the MTT assay using the approach described
in Section 3.2. Neutralization of each decontamination
liquid was attempted by dilution with hard water (400 ppm
as CaCO3) alone and by the addition of Dey and Engley
(D/E) neutralizing broth (solutions ranging from 50-95%
D/E broth) followed by dilution with hard water. Fifty
percent D/E broth was used for pH-amended bleach (3.0 mL
decontamination liquid + 3.0 mL neutralizer), 75% D/E
broth was used for 1% citric acid (1.5 mL decontamination
liquid + 4.5 mL neutralizer), and 75% D/E broth was used
for 732 ppm quaternary ammonium (1.5 mL decontamination
liquid + 4.5 mL neutralizer). Sodium carbonate only required
a 1:2 dilution of the decontamination liquid itself, with no
need for the neutralizer (3.0 mL decontamination liquid
+ 3.0 mL hard water). The neutralization method for each
decontamination liquid was generally selected based on the
lowest amount of D/E broth that needed to be added (if any)
in order to maintain >90% of the mean unexposed control
MDCK cell viability after diluting the neutralization solution
(with hard water) at a ratio of 1:2 (i.e., equal amounts of
neutralization solution and hard water were used). The
cytotoxicity test results are provided in Table 3-1 for the
decontamination liquids and the selected neutralization
approaches.
Table 3-1. Neutralized Decontamination Liquid Cytotoxicity Test Results
1% Citric Acid
75% D/E broth3
1:8
0.3336
0.3240
97.l
pH-Amended Bleach
50% D/E brothb
1:2
1.0299
1.0795
104.8e
732 ppm Quaternary
Ammonium
75% D/E brothc
1:2
1.0610
1.0216
96.3
8% Na2C03
No neutralizer (D/E broth)d
1:2
0.8761
0.8354
95.4
a 1.5 ml citric acid + 4.5 ml D/E broth.
b 3.0 ml pH-amended bleach + 3.0 ml D/E broth.
c 1.5 ml quaternary ammonium + 4.5 ml D/E broth.
d 3.0 ml Na2C03 + 3.0 ml hard water.
e Viability above 100% may reflect non-specific reduction of MTT to formazan from unknown coupon extract components.
-------
3.3 H5N1 Virus Recovery From Extraction Buffer,
Neutralized Decontamination Liquid, and Trough
Decontamination Liquid
The decontamination technology evaluation utilized test
coupons that were each spiked and dried as described
in Section 2.3 for persistence testing. For the evaluation
conducted at the lower temperature, the coupons were first
dried for one hour at room temperature, then placed in the
refrigerator for one hour before adding the decontamination
liquids (also placed in the refrigerator one hour prior to
testing). In the decontamination technology evaluations,
spiked test coupons and procedural blank coupons were
inverted (spiked surface down) and placed into separate
troughs for galvanized metal and separate vials for soil
each holding enough decontamination liquid to cover the
spiked surface of the coupon. At the end of the 10-minute
decontamination contact time, the coupons were removed,
neutralized in separate vials, extracted to recover H5N1 virus,
and extracts assayed as described in Section 2.3.
For neutralization to be acceptable, it should not interfere
with the methods used for extraction and quantification of the
virus, and the neutralization must be effective at inhibiting
the virucidal activity of the decontamination liquid. Three
method demonstration tests were conducted to determine the
applicability of each neutralization approach:
• H5N1 virus recovery from coupons using extraction
buffer
• H5N1 virus recovery from neutralized
decontamination liquid
• H5N1 virus recovery from trough decontamination
liquid.
Table 3-2. H5N1 Virus Recovery From Coupons Using Extraction Buffer
3.3.1 H5N1 Virus Recovery From Coupons Using
Extraction Buffer
The intent of these tests was to demonstrate the ability
to recover the H5N1 virus from coupons undergoing the
test procedures of decontamination and neutralization by
using only extraction buffer (PBS), essentially serving
as a recovery control. Spiked coupons (galvanized metal
and soil) were added to troughs/dishes containing PBS.
After a 10-minute contact time, the test coupons were
then removed and extracted and quantified as described in
Section 2.3.2. For galvanized metal, the trough solution was
also quantified to capture any virus washing into the trough
liquid; soil test coupons became a mixture with the PBS
in the vial and separate quantification of the trough liquid
was not applicable. These tests were conducted three times
(Trials A, B and C; each with three replicates) and the mean
H5N1 virus recoveries are presented in Table 3-2. Although
the recoveries were generally less than the performance
criterion of >25% of the spiked level, as discussed in
the test/QAplan1, adequate quantities of the H5N1 virus
remained and therefore decontamination efficacy (reported
as log reduction) could be accurately assessed. Note: EPA's
virucidal test guidance does not require a minimum percent
recovery, but rather a minimum virus recovery (TCID50) of
>104>12.
Trail A
Galvanized Metal (total; coupon + trough)
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil
Trial B
Galvanized Metal (total; coupon + trough)
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil
Trial C
Galvanized Metal (total; coupon + trough)
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil
Ifllllllfl IV". Vj i i f 1 1' fit ft K '1 1 1'?1; !? »isiisssssssssssssssif 8 \ 'A ii * ?!ftlIIIIIIIIIIIII|l ,''. f ;V1 */>;! •/! I (ft: if ^ Tii'n'?'".'!.; J
Wmwaw^mfem^ !•>:
5.01xl06 8.17x10= 13
4.39xl05 8.77
3.78xl05 4.52
5.01 x 106 2.99xl05 3.58
2.00xl06 5.40x10= 27
5.37x10= 26.9
3.16xl03 0.09
2.00xl05 3.16x10= 9.48
5.01 x 107 1.52xl07 30
9.49 x 106 19.0
5.70xl06 11.4
5.01xl07 1.75xl06 3.50
' The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the
mean of these percentages.
-------
3,3,2 H5N1 Virus Recovery From Neutralized
Decontamination Liquid
The intent of this test was to determine the ability of the
neutralization approach (as summarized in table 3-1) to
inhibit the virucidal activity of the decontamination liquid.
Briefly, spiked coupons (three for each material) were added
to the troughs (for galvanized metal) or vials (for soil) of
neutralized decontamination liquid (i.e., the neutralization
solutions of Table 3-1; subsequent dilution with hard water at
1:2 was only required for the MTT assay) for the 10-minute
contact time. The coupons were then removed and extracted
and the H5N1 virus quantified as described in Section 2.3.2.
For galvanized metal the H5N1 virus in the trough solution
was also quantified to account for any virus washing into
the trough liquid; soil test coupons became a mixture in
the vials with the addition of neutralized decontamination
liquid; since no troughs were used, a separate quantitation
of the trough liquid was not applicable. The mean TCID50 of
H5N1 virus recovered from the neutralized decontamination
liquids are presented in Table 3-3. Although the recoveries
were generally less than the performance criterion of >25%
of the spiked level (per the test/QA plan), an appreciable
amount of the H5N1 virus remained (6.92 x 104 to 5.96 x
106 mean TCID50) and therefore decontamination efficacy
could be accurately assessed. Note: EPA's virucidal test
guidance does not require a minimum percent recovery, but
rather a minimum virus recovery (TCID50) of >104 (so that an
adequate log reduction could be demonstrated).
3.3.3 H5N1 Virus Recovery From Trough
Decontamination Liquid
The intent of these tests was to determine if the H5N1 virus
remained in the decontamination liquid after the coupons
(galvanized metal only) were removed from the troughs.
Spiked galvanized metal coupons were placed in troughs of
decontamination liquid for the 10-minute contact time and
then removed. Given the possibility of the H5N1 virus being
dislodged from the coupon during decontamination, the
decontamination liquid in the trough was neutralized, and the
H5N1 virus in the trough liquid was quantified as described
in Section 2.3.2. The H5N1 virus recovered from the trough
decontamination liquid is presented in Table 3-4. In view
of the finding that the H5N1 virus was sometimes detected
in the trough decontamination solution, the quantification
of this residual liquid was incorporated into the actual
decontamination technology evaluation approach to ensure
the recovery of all of the H5N1 virus.
Table 3-3. H5N1 Virus Recovery From Neutralized Decontamination Liquid
•iiiS W!E*s*f?«*'K*f;r^ !! II
1% Citric Acid + 75% D/E broth"
Galvanized Metal (total; coupon + trough) 5.01
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil 5.01
pH-Amended Bleach + 50% D/E broth"
Galvanized Metal (total; coupon + trough) 2.00
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil 2.00
8% Na2C03 + No D/E broth"
Galvanized Metal (total; coupon + trough) 5.01
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil 5.01
732 ppm Quaternary Ammonium + 75% D/E broth"
Galvanized Metal (total; coupon + trough) 5.01
Galvanized Metal (from coupon)
Galvanized Metal (from trough)
Soil 5.01
I
x 107 5.96xl06 12
5.96 xlO6 11.9
0 0
x 107 2.60x10= 0.52
x 106 1.54xl05 7.6
1.51xl05 7.53
3.16xl03 0.09
x 106 6.96x10= 20.9
x 106 6.96 xlO4 1.4
6.96xl04 1.39
0 0
xlO6 6.92 xlO4 0.83
x 106 6.66x10= 13
5.96x10= 11.9
6.96xl04 0.83
xlO6 1.60 xlO6 19.1
1 The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the
mean of these percentages.
3 Unlike the cytoxicity test results of neutralized decontamination liquid shown in Table 3-1, subsequent dilution with hard water was not required
for this assay.
-------
Table 3-4. H5N1 Virus Recovery From Galvanized Metal Trough Decontamination Liquid
1% Citric Acid
5.01 x 107
0
pH-Amended Bleach
2.00x 106
3.16x 103
0.09
8% Na2C03b
Not tested
Not tested
Not tested
732 ppm Quaternary Ammonium
5.01x 106
0
0
1 The mean % virus recovery is determined by calculating the % virus recovery for each coupon (recovered virus/inoculum) and then computing the
mean of these percentages.
3 8% Na2C03 (which did not require D/E broth) for trough neutralization, was not tested.
3.4 Evaluation of Liquid Decontamination Technologies
3.4.1 Test Matrix
Log reductions in the H5N1 vims persistence were measured
on two materials: galvanized metal and soil. Based on
the relatively longer persistence times for the H5N1 virus
under cold temperatures and low RH (especially in the
absence of UV-A/B), the efficacy of decontamination liquids
was assessed at both room temperature (22 °C) and low
temperature (5 °C) for the 10 minute contact times. The
experimental treatments performed are shown in Table 3-5.
3.4.2 Test Results
The H5N1 virus recovery and log reduction in TCID50
for each decontamination liquid/environmental condition
combination are summarized in Tables 3-6 for galvanized
metal and Table 3-7 for soil. Table 3-6 and 3-7 also include
the number of replicates for positive controls (5), test
Table 3-5. Decontamination Technology Evaluation Matrix
coupons (5), laboratory blank (1), and procedural blank
(1). A summary of the log reductions obtained in all
decontamination liquid tests is provided in Table 3-8. For the
decontamination evaluations, the mean room temperature
was 23.31 °C with a range of 23.11 °C to 23.33 °C and the
mean low temperature was 5.34 °C with a range of 4.20 °C
to6.10°C.
Following a 10-minute contact time, 8% Na2CO3 and
732 ppm quaternary ammonium, reduced the H5N1 virus
on galvanized metal by less than 1.0 log in TCID50 at room
temperature and low temperature test conditions (Table 3-6).
When 1% citric acid was applied, mean log reductions in
H5N1 virus were 2.04 at room temperature and 2.13 at low
temperature. Only pH-amended bleach with a 10-minute
contact time, completely inactivated the H5N1 virus from
galvanized metal (no detectable H5N1 virus), with mean
log reductions of 7.1 at room temperature and 6.9 at the low
temperature.
^fi^fi^fi^«^«tf«i/«^
1% Citric Acid
pH-Amended Bleach
8% Na2C03
732 ppm Quaternary Ammonium
Galvanized metal
Galvanized metal
Soil
Soil
Galvanized metal
Galvanized metal
Soil
Soil
Galvanized metal
Galvanized metal
Soil
Soil
Galvanized metal
Galvanized metal
Soil
Soil
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
Room temperature
Low temperature
-------
Table 3-6. Decontamination Efficacy Against H5N1 Virus on Galvanized Metal
pH-Amended Bleach
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
Room Temperature
1% Citric Acid
Positive Control0
Test Coupond
Laboratory Blank6
Procedural Blank*
pH-Amended Bleach
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
8% Na2C03
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
732 ppm Quaternary Ammonium
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
2.00xl07
2.00x 107
0
0
7.94x 107
7.94xl07
0
0
7.94xl07
7.94x 107
0
0
7.94x 107
7.94x 107
0
0
5/5
5/5
0/1
0/1
5/5
0/5
0/1
0/1
5/5
5/5
0/1
0/1
5/5
5/5
0/1
0/1
9.86±0.98xl05
1.00±0.58x 105
ND
ND
2.76± 1.18x 107
ND
ND
ND
2.76± l.lSxlO7
3.71 ±0.96x 106
ND
ND
2.76± 1.18x 107
5.64± 1.58x 106
ND
ND
49.3 ±
0.50±
-
-
34. 8 ±
0.00 ±
-
-
34.8 ±
4.67 ±
-
-
34. 8 ±
7.10±
-
-
4.87
0.29 2.0 ±0.2
-
-
14.9
0.008 7.1 ±0.08
-
-
14.9
1.21h' 0.88±0.11h
-
-
14.9
1.99 0.70 ±0.12
-
-
Low Temperature
1% Citric Acid
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
1.28xl08
1.28x 108
0
0
5/5
5/5
0/1
0/1
5.83±2.95xl07
5. 49 ±4. 27 x 105
ND
ND
45.5 ±23.1
0.43 ± 0.33
-
-
-
2.1 ±0.4
-
-
5.01 x 107
5.01xl07
0
0
5/5
0/5
0/1
0/1
1.43 ±0.53 x 107
ND
ND
ND
28.5 ± 10.5
0.00 ± 0.008
6. 9 ±O.Qs
8% Na2C03
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01xl07
5.01 x 107
0
0
5/5
5/5
0/1
0/1
1.43±0.53xl07
2.19± 1.86x 106
ND
ND
28.5 ± 10.5
4.37 ±3.71
0.93 ± 0.35
732 ppm Quaternary Ammonium
Positive Control
Test Coupon
Laboratory Blank
Procedural Blank
5.01 x 107
5.01 x 107
0
0
5/5
5/5
0/1
0/1
1.43 ±0.53 x 107
6.05 ±2.70 x 106
ND
ND
28.5 ± 10.5
12.1 ±5.39
0.43 ± 0.29
a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicates.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls were inoculated, placed in PBS and exposed to the environmental condition, extracted after the 10-minute contact time. Only
one set of positive controls was used for each environmental condition and all four decontamination liquids.
d Test coupons were inoculated, exposed to the environmental condition and decontamination liquid for the 10-minute contact time.
e Laboratory blanks were not inoculated, extracted at time-zero.
f Procedural blanks were not inoculated, placed in PBS, exposed to the environmental condition, extracted after the 10-minute contact time.
8 A value of 1 TCID5o was used for non-detects (from the coupon and the trough liquid, which are summed to determine the total amount of virus)
in the calculation of % recovery and mean log reduction.
h Although H5N1 virus was recovered from all five replicate galvanized metal coupons, H5N1 virus was not detected from the associated trough
liquid (quantification of virus from the coupon and trough liquid is summed to determine the total amount of virus).
A value of 1 TCID50 was used for non-detects in the trough liquid for calculation of % virus recovery and mean log reduction.
ND = Not detected; the detection limit was 1.31 x 103 TCID50.
"-" Not applicable.
-------
Table 3-7. Decontamination Efficacy Against H5N1 Virus on Soil
ilil7<:»:^ |
W-, 11- W!!»:f '^^^^^^^ViitWfgillllST!.^.;;-.^^
MHlfw^
Room Temperature
1% Citric Acid
Positive Control0 2.00xl07 5/5
Test Coupond 2.00xl07 5/5
Laboratory Blank6 0 0/1
Procedural Blank' 0 0/1
pH-Amended Bleach
Positive Control 7.94xl07 5/5
Test Coupon 7.94xl07 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
8% Na2C03
Positive Control 7.94xl07 5/5
Test Coupon 7.94xl07 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
732 ppm Quaternary Ammonium
Positive Control 7.94xl07 5/5
Test Coupon 7.94xl07 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
3. 12 ±2. 80 x 106 15.62 ± 14.0
1.25 ±0. 78 x 105 0.63 ±0.39 1.5 ±0.4
ND
ND
4.27 ± 1.01 x 107 53.8 ±12.8
9. 97 ±3. 05 x 104 0.13 ±0.04 2.7 ± 0.2
ND
ND
4.27 ± 1.01 x 107 53.8 ±12.8
6.86 ±4. 97 x 106 8.64 ± 6.26 0.95 ± 0.49
ND
ND
4.27± l.Olx 107 53.8 ±12. 8
1.63 ± 0.43 x 107 20.6 ±5.36 0.43 ±0.11
ND
ND
Low Temperature
1% Citric Acid
Positive Control 1.28xl08 5/5
Test Coupon 1.28xl08 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
pH-Amended Bleach
Positive Control 5.01 x 107 5/5
Test Coupon 5.01 x 107 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
8% Na2C03
Positive Control 5.01 x 107 5/5
Test Coupon 5.01 x 107 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
732 ppm Quaternary Ammonium
Positive Control 5.01 x 107 5/5
Test Coupon 5.01 x 107 5/5
Laboratory Blank 0 0/1
Procedural Blank 0 0/1
2.83 ± 1.32 x 107 22. 14 ±10. 28
7.10±3.94x 105 0.55±0.31 1.7 ±0.3
ND
ND
2.16± 1.63x 107 43.1 ±32. 5
3.29 ± 1.68 x 104 0.07 ±0.03 2.9 ± 0.3
ND
ND
2.16± 1.63x 107 43.1 ±32. 5
1.15± 1.09x 107 22. 9 ±21. 8 0.42 ± 0.38
ND
ND
2.16± 1.63x 107 43.1 ±32. 5
8.09 ±3. 26 x 106 16.1 ±6. 51 0.45 ±0.17
ND
ND
a CPE detections: the numerator is the number of coupons with CPE detected and the denominator is the total number of replicate coupons.
b Data are expressed as mean + standard deviation as applicable.
c Positive controls were inoculated, placed in PBS and exposed to the environmental condition, extracted after the 10-minute contact time.
d Test coupons were inoculated, exposed to the environmental condition and decontamination liquid for the 10-minute contact time.
e Laboratory blanks were not inoculated, extracted at time-zero.
f Procedural blanks were not inoculated, placed in PBS and exposed to the environmental condition, extracted after 10-minute contact time.
ND = Not detected; the detection limit was 1.31 x 103 TCID5a
"-" Not applicable.
-------
None of the evaluated decontamination technologies
completely inactivated the H5N1 vims on soil (Table 3-7).
For 8% Na2CO3 and 732 ppm quaternary ammonium (at
room and the cold temperature), mean H5N1 log reductions
on soil were less than 1.0. When 1% citric acid was applied,
mean log reductions in H5N1 virus were 1.5 at room
temperature and 1.7 at low temperature. When pH-amended
bleach was used the mean H5N1 virus log reductions on soil
were 2.7 at room temperature and 2.9 at the low temperature.
Table 3-8. Summary of Decontamination Efficacy Against H5N1 Virus
Galvanized Metal
Room Temperature
Low Temperature
2.0 ±0.2
2.1 ±0.4
7.1 ±0.0b
6.9±0.0b
0.88±0.11C
0.93 ±0.35
0.70 ±0.12
0.43 ±0.29
Soil
Room Temperature 1.5 ± 0.4
Low Temperature 1.7 ± 0.3
2.7 ±0.2
2.9 ±0.3
0.95 ±0.49
0.42 ±0.38
0.43 ±0.11
0.45 ±0.17
1 Data are expressed as mean + standard deviation.
3 H5N1 virus was not detected from any of the replicate test coupons or associated trough liquid; a value of 1 TCID50 was used for these non-
detects in the calculation of the H5N1 virus mean log reduction in TCID50.
: H5N1 virus was not detected from the associated trough liquid; a value of 1 TCID50 is used for non-detects in the calculation of H5N1 virus
mean log reduction in TCID5o.
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4.0
Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were
performed in accordance with the test/QA plan1 and the
TTEP QMP2 for the persistence testing and decontamination
technology evaluation. QA/QC procedures are summarized
below. Some experimental procedures could not be
conducted in accordance with the test/QA plan; those
deviations are summarized in Section 4.5.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety
cabinets) used at the time of use 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., spectrophotometric absorbance, temperature, RH, and
contact time), will be conducted and reported in conjunction
with the virus persistence/decontamination testing associated
with freeze-dried vaccinia virus.
4.2.2 Technical Systems Audit
A technical systems audit was conducted on April 16, 2009.
The assessment addressed all steps from removal from the
cold storage (low temperature) followed by application of
the decontamination technologies, neutralization, extraction,
preparation of serial dilutions, and seeding onto cells. Only
steps missed were staining of plates and reading of final
plaques. All equipment used was within calibration and no
other items were noted. In addition, all known deviations
from the test procedures specified in the test/QA plan have
been documented in this listing of deviations in Section 4.5.
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 Quality Assurance/Quality Control Reporting
The audit described in Section 4.2.3 was documented
in accordance with the QMP2. 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 and decontamination
test 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 plan1 occasionally arose once
work in the laboratory was initiated; these deviations were
not expected to adversely affect data quality and included:
1. Soil and chicken feces were not autoclaved (avoiding
potential effects on the soil matrix, such as hydrolysis
of humic acids), but instead filtered through a
0.2 um filter during the extraction process to remove
bacteria (not viruses) from the supernatant that could
potentially interfere with virus quantification. Blanks
(non-inoculated coupons) confirmed that any naturally
occurring viruses did not interfere with the TCID50
assay (i.e., the TCID50 was generally not detected).
For soil, there was only one occasion (the 9-day test at
low temperature, high RH, no UV) where TCID50 was
detected in the procedural blank.
2. Unpainted concrete and soil coupons were too small to
spike with a multichannel pipette, so a single channel
pipette was used for these coupons. There were no
adverse impacts on the testing.
3. Regarding the assessment of potential cytotoxic effects
of materials extracts, that could potentially interfere
with the TCID50 assay, the cell viability performance
criterion of >90% of unexposed control cell viability
with dilution of 1:8 or less was not attained for soil;
however >90% of unexposed control cell viability
was obtained with a dilution of 1:16, which was
approved as an acceptable level of dilution by the EPA
Task Order Project Officer. As noted in Section 2.2,
although an increased level of dilution was needed
for the MTT assay with soil, the TdD50 assay was
not affected as cytopathic effects were generally not
observed in the MDCK cells exposed to any dilution
of soil extract. There were no adverse impacts on the
testing.
4. Alternative approaches were used for generating high
RH and low temperature environmental conditions
than proposed in the test/QA plan1. Specifically,
instead of using a nebulizer to generate a high RH at
the room temperature condition, the coupons were
placed in a glove box pre-humidified by using an
ultrasonic-fogger. The moist test material (i.e., soil
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and chicken feces) maintained a high RH level. For
conducting low temperature test, rather than running
tubing from a re-circulating chiller through the glove
box, testing was conducted inside a refrigerator. When
testing at a low temperature with no UV the coupons
were placed inside a Lock&Lock™ plastic storage
container which was placed in the refrigerator. When
testing at a low temperature with UV-A/B the coupons
were placed directly into a refrigerator modified to
include glove ports. There were no adverse impacts on
the testing.
5. Continuous monitoring of temperature and RH was
inadvertently not conducted during the H5N1 virus
persistence testing at the room temperature, low RH,
no UV environmental condition and during the initial
seven hours of the 1-day test at the low temperature,
low RH, UV-A/B environmental condition. No unusual
events occurred that would lead one to expect that the
temperature and RH of these tests deviated from those
of comparable tests.
6. Temperature and RH inside the test chamber were
found to be difficult to control (especially when trying
to test non-ambient conditions over relatively long
periods of time, i.e. days) and were often outside of the
target environmental conditions (±2 °C for temperature
and ±10% for RH), as documented in Section 2.4.
However, these deviations from target environmental
conditions do not invalidate the TCID50 data. The
actual (measured) temperature and RH data are all
reported.
7. The performance criteria of no observed CPE in
MDCK cells for laboratory and procedural blanks
often failed for chicken feces; however laboratory
and procedural blanks with CPE were not rejected
because they were attributed to the test material itself.
Relatively low detections of TCID50 (i.e., <3.16 x
103 TdD50) in chicken feces test coupons were often
nagged as not being detected above the associated
blank of 3.16 x 103 TCID50 (i.e., it could not be
determined if the CPE were attributed to the H5N1
virus or simply the chicken feces).
8. Contrary to the method development tests, most of
the positive control virus recoveries were below
the performance criterion (>20% and <120% virus
recovery). However, even the lowest percent virus
recovery (0.8%) had a mean of 2.54 x 105 TdD50
recovered, which is an appreciable amount of virus
to assess persistence. EPA's virucidal test guidance
does not require a minimum percent recovery, but
rather a minimum virus recovery (TCID50) of >104. It
is possible that having a lower virus liter initially may
make the virus seem less persistent than it would have
been with a higher initial titer.
9. Regarding the decontamination technology evaluation,
the positive controls (at the 10-minute contact time)
were exposed to PBS rather than being exposed only to
air, which better mimicked the actual application of the
decontamination liquid.
10. Although the H5N1 virus recoveries from neutralized
decontamination liquid and extraction buffer were
generally <25%, the H5N1 virus recoveries were
>6 x 104 TCID50, which is an appreciable amount of
virus to assess decontamination efficacy. Note: EPA's
virucidal test guidance does not require a minimum
percent recovery, but rather a minimum virus recovery
(TCID50) of >104. The H5N1 virus was detected
in the trough decontamination liquid, such that the
H5N1 virus recovery in the actual decontamination
technology evaluation was quantified from the coupon
and the liquid in the trough (for galvanized metal
only) to ensure that all of the viable H5N1 virus was
quantified.
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5.0
Summary
5.1 H5N1 Virus Persistence
The persistence of the H5N1 vims (A/Vietnam/1203/04) was
investigated on four materials (chicken feces, galvanized
metal, glass, and soil) under five environmental conditions
(each condition consisted of various combinations of
temperature, RH, and exposure to UV-A/B radiation [to
simulate sunlight]). Under room temperatures at both low
and high RH (no UV), the H5N1 virus persisted for less
than two days on chicken feces, galvanized metal, glass, and
soil. At low temperatures under both low and high RH (no
UV) the H5N1 virus persisted for at least four days on all
materials. The H5N1 virus was detected following exposure
to the low temperature, low RH, no UV environmental
condition after 13 days on galvanized metal, glass, and soil.
The H5N1 virus was detected on chicken feces, glass, and
soil following exposure to the low temperature, high RH,
no UV environmental condition for nine days. Testing was
not conducted for durations longer than 13 days for any of
the environmental conditions; the duration of H5N1 virus
persistence may exceed 13 days, especially on galvanized
metal and glass under the low temperature, low RH, no UV
environmental condition.
With continuous UV-A/B exposure to simulate sunlight
and under a low temperature and low RH environmental
condition, the H5N1 virus persisted less than 48 hours
on galvanized metal and glass. H5N1 persisted 96 hours
on soil and chicken feces, although for chicken feces, the
H5N1 level recovered from the test coupons could not be
distinguished from the interference of the test material itself.
5.2 H5N1 Virus Liquid Decontamination
Four decontamination liquids (1% citric acid, pH-amended
bleach, 732 ppm quaternary ammonium, and 8% Na2CO3)
prepared with hard water were applied to galvanized metal
and soil coupons inoculated with the H5N1 virus, and tested
at room and low temperature conditions for 10-minute
contact times. The decontamination technology evaluation
indicated that only pH-amended bleach was effective at
completely inactivating the H5N1 virus (i.e., the H5N1
virus was not detected), and this result was only obtained
for galvanized metal (at both room and low temperatures).
For soil, pH-amended bleach induced a mean H5N1 virus
log reductions in TCID50 of 2.7 at room temperature and 2.9
at low temperature. The mean H5N1 virus log reductions
in TdD50 associated with 1% citric acid ranged from 1.5
(with soil at room temperature) to 2.1 (with galvanized metal
at low temperature). The mean log reductions in the H5N1
virus, determined for 732 ppm quaternary ammonium and
8% Na2CO3, were all less than 1.0 for both temperature
conditions tested.
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6.0
References
1. Technology Testing and Evaluation Program Test/QA Plan for Persistence Testing of Avian Influenza
Viruses on Outdoor Materials, Version 1, Prepared for EPA by Battelle, Columbus, Ohio,
March 2007.
2. Quality Management Plan (QMP) for the Technology Testing and Evaluation Program (TTEP),
Version 1, Prepared for EPA by Battelle, Columbus, Ohio, January 2005.
3. Finney, D. J. Assays Based on Quantal Responses. In: Statistical Methods in Biological Assays, 3rd
ed. Macmillan Publishing Co., Inc., New York, p. 394-398. 1978.
4. Battelle, BBRC Method No. 125/Microbiology, Method for the Determination of the Median Tissue
Culture Infectious Dose (TCID50) for a Viral Sample. Battelle Proprietary.
5. Centers for Disease Control and Prevention (CDC). Guidance & Recommendations, Interim
Guidance for Protection of Persons Involved in U.S. Avian Influenza Outbreak Disease Control and
Eradication Activities, http://www.cdc.gov/flu/avian/professional/protect-guid.htm.
6. Minis, P.M., III. Avian Influenza and UV-B Blocked by Biomass Smoke. Environmental Health
Perspectives, 2005 113(12) A806-A807.
7. Jeanmougin, M. and J. Civatte. Dosimetry of Solar Ultraviolet Radiation. Daily and Monthly
Changes in Paris. [Article in French] AnnDermatol Venereol, 1987 (114):671-676.
8. Kolari, P. J., J. Lauharanta, and M. Hoikkala. Midsummer Solar UV-Radiation in Finland Compared
with the UV-Radiation from Phototherapeutic Devices Measured by Different Techniques.
Photodermatol, 1986 (3):340-345.
9. Balasaraswathy, P., U. Kumar, C.R. Srinivas, and S. Nair. UVA and UVB in Sunlight, Optimal
Utilization of UV Rays in Sunlight for Phototherapy. Indian J Dermatol Venereol Leprol, 2002
(68): 198-201.
10. McNamara, A.E. and W.R. Hill. UV-B Irradiance Gradient Affects Photosynthesis and Pigments but
Not Food Quality of Periphyton. Freshwater Biology, 2000 (43):649-662.
11. Diffey, B.L. Sources and Measurement of Ultraviolet Radiation. Methods, 2002 (28):4-13.
12. EPA, Antimicrobial Science Policies, Disinfectant Technical Science Section, available online at:
http://www.epa.gov/oppad001/sciencepolicv.htm.
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Appendix A:
H7N2 Virus Testing
In an effort to generate paired persistence and
decontamination efficacy data for highly pathogenic avian
influenza virus (i.e., H5N1 virus, strain A/Vietnam/1203/04)
and low pathogenic avian influenza virus (i.e., H7N2 virus
strain A/chicken/Mary land/Minh Ma/04), preliminary
research was also conducted with the H7N2 virus.
To improve comparability of the test results for both viruses,
it was desired to quantify both viruses using the same
approach (i.e., the TdD50 assay). However, initial efforts at
propagating the H7N2 virus resulted in virus concentrations
that were less than the target titer of 1 x 107 TCID50 (using
MDCK cells for virus quantitation, as with the H5N1
virus). Re-propagation of the H7N2 virus continued to yield
apparently low liters; however, this was eventually attributed
to the use of a mammalian cell line (MDCK) to quantify
the H7N2 virus. Attempts were thus made to quantify the
H7N2 virus using chicken embryo kidney (CEK) cells to
conduct the TCID50 assay. Initial results indicated that CEK
cells may be used to quantify the H7N2 virus and yielded
results indicating that the propagated H7N2 virus stock had
a titer of 1.26 x 107 TCID50/mL. However unacceptably high
variability of this assay was obtained with repeated assays.
Research was conducted to evaluate the effect of different
aged CEK cells with the TCID50 assay to reduce the assay
variability, but the titer results remained too variable to draw
conclusions (see Table A-l). It was determined that additional
research was needed to develop a reliable CEK assay for
H7N2 virus, which was beyond the scope of the current
project.
Table A-l. H7N2 Virus liters Obtained Using Chicken Embryo Kidney Cells3
4/13/07 H7N2 Stock (l:500)b
1.26 x 106
5.01xl05
1.26 x 106
4/13/07 H7N2 Stock (l:1000)c
2.00x 104
2.00x 105
3.16x 105
4/13/07 H7N2 Stock (l:5000)d
5.01 x 103
3.16x 105
5.01 x 105
5/4/07 H7N2 Stock
2.00 x 103
1.26x10=
2.00x 105
5/25/07 H7N2 Stock6
2.00x 103
2.00x 105
2.00x 106
a The results of using the fresh (plated within 48 hours of receipt from vendor) CEK cells (7/31/07 titer) and the aged (plated after 48 hours after
receipt from vendor) CEK cells (8/1/07 titer) were compared with initial titer results; all results were based on the fifth day of post-infection.
b 1:500 = initial 1:10 dilution of H7N2 virus stock (0.01 ml virus stock + 0.09 ml diluent), final 1:50 dilution of initial dilution (0.03 ml 1:10
virus dilution + 1.47 ml diluent) for a 1:500 quantity of virus needed to infect eggs.
c 1:1000 = initial 1:100 dilution of H7N2 virus stock (0.01 ml virus stock + 0.99 ml diluent), final 1:10 dilution of initial dilution (0.15 ml
1:10 virus dilution + 1.35 ml diluent) for a 1:1000 quantity of virus needed to infect eggs.
d 1:5000 = initial 1:100 dilution of H7N2 virus stock (0.01 ml virus stock + 0.99 ml diluent), final 1:50 dilution of initial dilution (0.03 ml
1:10 virus dilution + 1.47 ml diluent) for a 1:5000 quantity of virus needed to infect eggs.
e Titered at 1.26 x 107 TCID50/ml_ on 6/28/07; cells plated two days after receiving.
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