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technical BR
Results From Persistence Testing of Biological Agents
Under Various Conditions
EPA investigates the persistence of biological agents under various conditions of
relative humidity, temperature, and light
Introduction
U.S. EPA's Homeland Security Research Program
(HSRP) develops products based on scientific
research and technology evaluations. Our products
and expertise are widely used in preventing,
preparing for, and recovering from public health and
environmental emergencies that arise from terrorist
attacks. Our research and products address
biological, radiological, or chemical contaminants that
could affect indoor areas, outdoor areas, or water
infrastructure. HSRP provides these products,
technical assistance, and expertise to support EPA's
roles and responsibilities under the National
Response Framework, statutory requirements, and
Homeland Security Presidential Directives.
Because of their potential use as weapons of mass
destruction, biological agents are a significant terrorist
threat. Once released, agents such as bacteria and
viruses can cause disease or death in humans,
animals, and plants by spreading through air, water
distribution systems, and the food supply. A major
attack could cause many casualties and interrupt vital
civilian and government operations.
Data on how long and under what conditions agents
remain viable or active influence many aspects of
planning, response, containment, and recovery from
biological incidents.
Following a contamination event, environmental
conditions that might decrease the number of viable organisms could be implemented prior to
decontamination efforts. Such pre-treatment could potentially reduce the risks of exposure,
lower the costs of cleanup, and shorten the time before re-use of a facility or an outdoor area.
Persistence testing of biological agents by EPA1
During persistence testing, standardized samples of materials (coupons) are spiked with a
biological agent or a non-pathogenic surrogate. For different intervals, coupons are exposed to
various experimental conditions such as changes in temperature, relative humidity (RH), or
periods of simulated sunlight. Coupons are then tested for the presence of viable organisms.
Testing and analysis of data follow quality assurance protocols.
The following are important environmental factors that affect persistence, which is the length of
time a microorganism or biological agent remains viable on a surface:
• Characteristics and amount of the biological agent
• Relative humidity (RH)
• Temperature (TEMP)
Note that the studies on persistence of various organisms involved differing coupon materials. These studies
were performed to gather specific information on organism persistence, not to cross-compare study results. For
example, the avian influenza virus study involved soil and chicken feces because these are environments of
concern for that virus.
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• Exposure to simulated sunlight
• Type of coupon material
• Porosity of material surface
The following disease causing microorganisms were studied:
Microorganisms
Bacillus anthracis
Brucella suis
Francisella tularensis
Highly pathogenic avian influenza (H5N1)
Vaccinia virus, the surrogate for Variola major
Yersinia pestis
Diseases
anthrax
brucellosis
tularemia
HPAI H5N1 influenza
smallpox
plague
As shown below, investigators found that increased temperature (TEMP), increased RH, and
exposure to simulated sunlight (0) [ultraviolet light with wavelengths of 280 to 400 nanometers
(UV-A/B)], tended to decrease the persistence of some biological agents. Generally, these
environmental conditions were found to decrease persistence of agents on most of the materials
tested. (See references [1,2,3,4,5,6] for full reports that have details on conditions, agents, and
materials.)
Summary of Tested Environmental Conditions Resulting in a Decreased Number of
Viable Organisms
Biological Agent
Conditions That Decreased the Number of Viable Organisms
Highly Pathogenic Avian Influenza
Exposing to simulated sunlighta at 22°C
Increasing temperature from 4°C to 22°Cb
-o-
TEMP
Vaccinia Virus (freeze-dried) [2]
Increasing relative humidity to > 70% at 22°C
•o-
RH
Vaccinia Virus [3]
Increasing relative humidity to > 70% at 30°C
•o-
RH
Brucella suis [6]
Exposing to simulated sunlightc at 22°C
0
a UV-A/B - exposure was continuous
b Persistence was higher at low temperature (4° C), than at moderate temperature (22° C) at both low and high relative humidity
c UV-A/B - exposure was 12 hours on and 12 hours off
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Summary of Major Results from Persistence Testing
Highly Pathogenic Avian Influenza (H5N1) [1]
Highly pathogenic avian influenza (H5N1) virus [influenza AA/ietnam/1203/2004 (H5N1,
clade 1) was spiked on coupons of glass, galvanized metal, topsoil, and chicken feces.
Environmental conditions were defined as low temperature (4°C), room temperature (22°C), low
RH (40%), high RH (80%), and exposure/no exposure to UV-A/B.
At low temperature, low RH, and no UV-A/B, viable H5N1 was recovered from glass, galvanized
metal, and topsoil on Day 13 of the 13 day test runs.
• Room temperature (at low and high RH and no UV-A/B exposure) decreased H5N1
persistence on glass, galvanized metal, topsoil, and chicken feces to less than 2 days
• UV-A/B decreased H5N1 persistence at low temperature and low RH to 4 or fewer days
on glass, galvanized metal, and topsoil
Vaccinia Virus (Freeze-Dried) [2]
Vaccinia virus is used in research as a surrogate for Variola major, the virus which causes
smallpox. One strain of vaccinia is used in the live vaccine against smallpox. Vaccinia was
freeze-dried as part of the persistence investigation to emulate the conditions under which the
variola virus might be shipped as part of a bioterrorism incident.
Vaccinia virus (ATCC ® VR119) was spiked on coupons of glass, galvanized metal, painted
cinder block, and industrial carpet. The coupons were frozen overnight to prepare prior to being
freeze-dried. Environmental conditions were defined as low temperature (10°C), room
temperature (22°C), low RH (20%), and high RH (70%).
At low temperature and low RH, viable vaccinia virus was recovered from all materials on Day
56 of the 56 day test runs.
At room temperature and low RH, viable vaccinia virus was recovered from glass, galvanized
metal, and painted cinder block on Day 42 of the 56 test runs.
• High RH at room temperature decreased vaccinia virus persistence on glass, galvanized
metal, painted cinder block, and industrial carpet to less than 3 days
Vaccinia Virus [3]
Vaccinia virus (ATCC® VR119), used as a surrogate for the smallpox virus Variola major, was
spiked on coupons of painted cinder block and galvanized metal ductwork. Environmental
conditions were defined as ambient RH (20°C and 40 to 70% RH), high temperature/low RH
(30°C and < 40% RH), and high temperature/high RH (30°C and > 70% RH).
At high temperature/low RH, viable vaccinia virus was recovered from galvanized metal and
painted concrete on Day 14 of the 14 day test runs.
• High temperature/high RH decreased vaccinia virus persistence to 9 days on painted
concrete
• High temperature/high RH decreased vaccinia virus persistence to 3 days on galvanized
metal
Elevated RH appeared to decrease the persistence of vaccinia virus. Other modest effects from
temperature differences might have been masked by the large effects of RH, however.
Francisella tularensis and Yersinia pestis [4]
Francisella tularensis LVS (Battelle culture: OSU FTL361) and Yersinia pestis CO-92 (Battelle
culture: M-YPO166) were spiked on coupons of aluminum, industrial carpet, painted joint tape
May 2011 EPA/600/S-11/005
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paper, and computer keyboard keys. Environmental conditions were 35 to 45% RH and 22°C
for these persistence tests.
• Viable F. tularensis was recovered from computer keys on Day 7 of the 7 day test runs
• No viable F. tularensis was recovered after Day 1 on aluminum, industrial carpet, and
painted joint tape paper
• Viable Y. pestis was recovered from aluminum and painted joint tape paper on Day 7 of
the 7 day test runs
• Viable Y. pestis was recovered from computer keyboards keys on Day 3 and from
industrial carpet at 8 hours
Bacillus anthracis [5]
Bacillus anthracis Ames and Bacillus subtilis (ATCC® 19659) were spiked on coupons of glass,
bare pine wood, unpainted concrete, and topsoil. The bacteria were exposed to 12 hours of UV-
A/B alternating with 12 hours of darkness. No clear pattern of UV-A/B exposure times and
decreased viability was evident with all materials. Some viable spores of both B. anthracis and
B. subtilis were recovered at 56 days on all materials.
• UV-A/B decreased the quantity of viable B. anthracis and B. subtilis recovered from
glass by about 5 Iog10 after 672 exposure hours (56 days)
Brucella suis [6]
Brucella suis biotype I (Battelle BRU163) was spiked on coupons of aluminum, glass, bare pine
wood, unpainted concrete, and topsoil. Not all materials were tested under all conditions.
Environmental conditions were defined as low temperature (4°C or 7°C), moderate temperature
(22°C), and exposure/no exposure to UV-A/B. Relative humidity was ambient, averaging from
39 to 59%.
At low and moderate temperature, without exposure to UV-A/B, viable B. suis was recovered
from aluminum, glass, and topsoil on Day 28 of the 28 day test runs (at moderate temperature
viable B. suis was not recovered from topsoil at day 21 or day 28).
At low and moderate temperature, with exposure to UV-A/B, B. suis was recovered from topsoil
on the Day 14 of the 14 day test runs.
• UV-A/B at moderate temperature decreased the persistence of B. suis, which was
recovered from glass on day 1 and from aluminum on Day 7 of the 14 day test runs
Technology Evaluation Reports Referenced
[1] Choi, Y., Rogers, J., Chappie, D., and Wood, J. 2009. Highly Pathogenic Avian Influenza
H5N1 Virus Persistence Testing and Evaluation of Liquid Decontamination Technologies.
Investigation and Technology Evaluation Report. Washington, D.C.: U.S. Environmental
Protection Agency. EPA/600/R-09/054.
[2] Choi, Y., Shaw, M., Rogers, J., Chappie, D., Taylor, M., Riggs, K., Wllenberg, Z., and Wood,
J. 2009. Freeze-Dried Vaccinia Virus Persistence Testing and Liquid Decontamination
Technology Evaluation. Washington, D.C.: U.S. Environmental Protection Agency. EPA/600/R-
09/139.
[3] Stone, H., Rogers, J., Fleming, E., Choi, Y., Waugh, J., Richter, W., Taylor, M., Riggs, K.,
Wllenberg, Z., Krile, R., and Ryan, S. 2006. Impact of Temperature and Humidity on the
Persistence of Vaccinia Virus and Ricin Toxin on Indoor Surfaces. Washington, D.C.: U.S.
Environmental Protection Agency. EPA/600/R-08/002.
May 2011 EPA/600/S-11/005
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[4] Ryan, S. 2010. Persistence Testing and Evaluation of Fumigation Technologies for
Decontamination of Building Materials Contaminated With Biological Agents. Washington, D.C.
U.S. Environmental Protection Agency. EPA/600/R-10/086.
[5] U.S. Environmental Protection Agency. 2010. Investigation of Simulated Sunlight in the
Inactivation ofB. anthracis and B. subtilis on Outdoor Materials. Washington, D.C.: U.S.
Environmental Protection Agency. EPA/600/R-10/048.
[6] U.S. Environmental Protection Agency. 2010. Persistence Testing ofBrucella suis on
Outdoor Materials. Investigation Report. Washington, D.C.: U.S. Environmental Protection
Agency. EPA/600/R-10/026.
For more information, visit the EPA Web site at www.epa.gov/nhsrc.
Technical Contacts: Joseph Wood (wood.joe@epa.gov)
Shawn Ryan (ryan.shawn@epa.gov)
General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov)
May 2011 EPA/600/S-11/005
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