Inactivation of Poxviruses by Upper-Room UVC Light in a Simulated Hospital Room Environment

Abstract

In the event of a smallpox outbreak due to bioterrorism, delays in vaccination programs may lead to significant secondary transmission. In the early phases of such an outbreak, transmission of smallpox will take place especially in locations where infected persons may congregate, such as hospital emergency rooms. Air disinfection using upper-room 254 nm (UVC) light can lower the airborne concentrations of infective viruses in the lower part of the room, and thereby control the spread of airborne infections among room occupants without exposing occupants to a significant amount of UVC. Using vaccinia virus aerosols as a surrogate for smallpox we report on the effectiveness of air disinfection, via upper-room UVC light, under simulated real world conditions including the effects of convection, mechanical mixing, temperature and relative humidity. In decay experiments, upper-room UVC fixtures used with mixing by a conventional ceiling fan produced decreases in airborne virus concentrations that would require additional ventilation of more than 87 air changes per hour. Under steady state conditions the effective air changes per hour associated with upper-room UVC ranged from 18 to 1000. The surprisingly high end of the observed range resulted from the extreme susceptibility of vaccinia virus to UVC at low relative humidity and use of 4 UVC fixtures in a small room with efficient air mixing. Increasing the number of UVC fixtures or mechanical ventilation rates resulted in greater fractional reduction in virus aerosol and UVC effectiveness was higher in winter compared to summer for each scenario tested. These data demonstrate that upper-room UVC has the potential to greatly reduce exposure to susceptible viral aerosols. The greater survival at baseline and greater UVC susceptibility of vaccinia under winter conditions suggest that while risk from an aerosol attack with smallpox would be greatest in winter, protective measures using UVC may also be most efficient at this time. These data may also be relevant to influenza, which also has improved aerosol survival at low RH and somewhat similar sensitivity to UVC.

Full text

Inactivation of Poxviruses by Upper-Room UVC Light in a
Simulated Hospital Room Environment
James J. McDevitt
1
, Donald K. Milton
1,2
*, Stephen N. Rudnick
1
, Melvin W. First
1
1Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts, United States of America, 2Department of Work Environment, University
of Massachusetts Lowell, Lowell, Massachusetts, United States of America
Abstract
In the event of a smallpox outbreak due to bioterrorism, delays in vaccination programs may lead to significant secondary
transmission. In the early phases of such an outbreak, transmission of smallpox will take place especially in locations where
infected persons may congregate, such as hospital emergency rooms. Air disinfection using upper-room 254 nm (UVC) light
can lower the airborne concentrations of infective viruses in the lower part of the room, and thereby control the spread of
airborne infections among room occupants without exposing occupants to a significant amount of UVC. Using vaccinia
virus aerosols as a surrogate for smallpox we report on the effectiveness of air disinfection, via upper-room UVC light, under
simulated real world conditions including the effects of convection, mechanical mixing, temperature and relative humidity.
In decay experiments, upper-room UVC fixtures used with mixing by a conventional ceiling fan produced decreases in
airborne virus concentrations that would require additional ventilation of more than 87 air changes per hour. Under steady
state conditions the effective air changes per hour associated with upper-room UVC ranged from 18 to 1000. The
surprisingly high end of the observed range resulted from the extreme susceptibility of vaccinia virus to UVC at low relative
humidity and use of 4 UVC fixtures in a small room with efficient air mixing. Increasing the number of UVC fixtures or
mechanical ventilation rates resulted in greater fractional reduction in virus aerosol and UVC effectiveness was higher in
winter compared to summer for each scenario tested. These data demonstrate that upper-room UVC has the potential to
greatly reduce exposure to susceptible viral aerosols. The greater survival at baseline and greater UVC susceptibility of
vaccinia under winter conditions suggest that while risk from an aerosol attack with smallpox would be greatest in winter,
protective measures using UVC may also be most efficient at this time. These data may also be relevant to influenza, which
also has improved aerosol survival at low RH and somewhat similar sensitivity to UVC.
Citation: McDevitt JJ, Milton DK, Rudnick SN, First MW (2008) Inactivation of Poxviruses by Upper-Room UVC Light in a Simulated Hospital Room
Environment. PLoS ONE 3(9): e3186. doi:10.1371/journal.pone.0003186
Editor: Matthew Baylis, University of Liverpool, United Kingdom
Received March 28, 2008; Accepted August 19, 2008; Published September 10, 2008
Copyright: ß2008 McDevitt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grant R21AI053522 ‘‘Prevention of airborne smallpox transmission’’ and by NIEHS Center grant P30ES000002.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Donald_Milton@uml.edu
Introduction
Smallpox (variola major) is a high priority bioterrorist threat
agent, according to the Centers for Disease Control and
Prevention and Department of Homeland Security, which can
be easily transmitted from person to person, result in high
mortality rates, might cause public panic and social disruption,
and require special action for public health preparedness (http://
www.bt.cdc.gov/agent/agentlist-category.asp). Airborne spread
via respiratory droplet nuclei has been identified as a potential
contributing mode of transmission for smallpox[1,2] and preven-
tion of transmission by vaccination will likely be delayed until
public health authorities become aware of the outbreak and
initiate a vaccination program. In the early phases of such an
outbreak, significant secondary transmission of smallpox will take
place especially in locations where infected persons may
congregate, such as hospital emergency rooms. Therefore, public
health measures in addition to vaccination are needed.
Hospitals limit aerosol disease transmission in indoor spaces by
reducing the concentration of airborne microorganisms through
dilution ventilation. However, these measures are largely imprac-
tical beyond a limited number of respiratory isolation rooms due
to the large amounts of air exchange needed to significantly reduce
the threat of infection and are therefore costly in terms of heating
and cooling these large amounts of air. The high ventilation rates
required for respiratory isolation rooms are not routinely used in
emergency departments and waiting areas. With air disinfection,
costs are reduced since air does not have to be removed from
occupied spaces to remove potential infectious agents. Disinfection
using high-efficiency filtration to significantly reduce the threat of
airborne infection can be effective but requires more powerful fans
beyond what currently exist in the majority of public buildings and
also require additional energy consumption. Air disinfection using
upper-room 254 nm (UVC) light can lower the airborne
concentrations of infective organisms in the lower part of the
room, and thereby control the spread of airborne infections among
room occupants without exposing occupants to a significant
amount of UVC.[3–5] Upper-room UVC systems do not require
modification to ventilation systems, are low maintenance, and
relatively easy to install.[6,7] The use of upper-room UVC is also
economical. For example, the 25-watt lamps used as part of our
study would cost just over $40 per year assuming an electrical cost
of $0.20 per kilowatt-hour. Some hospitals currently employ
upper-room UVC for this purpose in their emergency depart-
ments (e.g. Brigham and Women’s Hospital, Boston, MA), but its
effectiveness against viral aerosols is not well established.
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Inactivation of microorganisms using UVC is often assumed to
follow a first-order decay with a susceptibility parameter Z = ln(1/
f) / D, where (f= organism fractional survival and D = UV dose,
where dose is the product of UV fluence rate –expressed as power
per cross sectional area—and exposure time, for example mJ/cm
2
.
Using a one-pass UVC exposure chamber, however, we have
shown that vaccinia virus (a surrogate for variola major) is
susceptible to UVC and that the susceptibility varies as a function
of dose and relative humidity (RH).[8] In these dose-response
experiments the fluence rate and exposure time, and therefore,
dose were carefully controlled. Thus, in each experiment, all
viruses received the same dose and we determined susceptibility to
UVC by varying dose over several experiments. In an actual room
using upper-room UVC, the UVC fluence rate varies even within
the upper-room, and the time spent in the upper-room varies from
particle to particle. Therefore, the dose for each viral particle
depends on the path that the particle travels. With perfect mixing,
particle doses would be exponentially distributed. In the case of
imperfect mixing, computational fluid dynamic (CFD) models
should be capable of describing the more complex distribution of
doses that would result. Then, using the pattern of UVC
susceptibility we previously reported, it should be possible to
estimate the net effectiveness of upper-room UVC. However,
given the complexity of UVC susceptibility that we previously
described combined with the complexity of CFD models,
empirical data are needed. We report experiments designed to
measure the effectiveness of upper-room UVC under simulated
real world conditions including the effects of convection,
mechanical mixing, temperature and relative humidity (RH).
Results
Decay
The environmental conditions within the chamber during decay
experiment were maintained at 2063uC and 50610% RH. The
results of chamber decay experiments performed with background
decay, without heat boxes, and with heat boxes are shown either
without the ceiling fan operating (Figure 1a) or with the ceiling fan
operating (Figure 1b). The exponential regression model fits the
data reasonably well. The rate constant shown in these equations
can be interpreted as the effective air exchange rate for the
chamber expressed in units of air changes per hour (ACH). Based
on a model for a chamber in which the air is perfectly mixed, the
effective air exchange rate is equal to the amount of virus-free
dilution air that would be needed to provide the same reduction of
virus concentration that was actually measured. The background
decay rate reflects the decrease in infective viruses due to the
exhaust airflow required to maintain negative pressure within the
chamber, as well as any physical and non-UVC-related biological
decay of the virus aerosol.
Virus reduction due to upper-room UVC is equal to the
effective air exchange rate with the UVC turned on minus the
effective air exchange rate with the UVC turned off (i.e. the
background decay rate). This difference, which is usually referred
to as equivalent air changes per hour (ACH
UVC
), is summarized in
Table 1. Overall, the rate of reduction of vaccinia virus increased
over the background as the amount of natural convection
increased; mixing by the ceiling fan overwhelmed natural
convection effects and markedly increased virus inactivation.
When the ceiling fan was not operating, the ACH
UVC
increased
by 7 ACH above background when viruses were dispersed at 37uC
(body temperature). When additional convective currents were
added to the room by the addition of two heat boxes (equivalent to
the heat generated by two people) the ACH
UVC
increased by 16
ACH. When the ceiling fan was in operation the ACH
UVC
increased to greater than 87 ACH and there was no discernable
effect attributable to the heat boxes.
Steady State
The average concentration of vaccinia aerosols during steady state
conditions with UVC off ranged from 1500 to 27000 pfu/m
3
.One
experiment(summer conditions with 2 ACH and 4 UVC fixtures), in
which the initial concentration before the UVC was turned on
(1500 pfu/m
3
) was much lower than any of the other experiments
was not used in our analysis, because the initial concentration was
too low to accurately measure .85% reductions in concentration.
The geometric mean vaccinia concentrations without UVC for the
experiments used in the analysis were 3400 (95% confidence interval
2600 to 4300) pfu/m
3
under summer conditions and 7800 (CI 5800
to 10000) pfu/m
3
under winter conditions. With UVC on, the
geometric mean concentrations were 570 (CI 430 to 770) pfu/m
3
in
the summer and 110 (CI 79 to 150) pfu/m
3
in the winter
experiments. The experiments under summer conditions showed
stronger time trends in aerosol concentrations and greater variability
between experimental replicates (i.e. steady state was difficult to
achieve even without the UVC fixtures). The fraction of infectious
virus remaining (ratio of the concentration of virus at steady state
with upper-room UVC on to that measured under steady state
conditions without UVC) is shown in Table 2 for the various
combinations of tested conditions: two ventilation rates (2 and 6
ACH), numbers of UVC fixtures (1 or 4 fixtures) and seasonal
conditions (summer and winter). Equivalent air changes due to UVC
under steady state conditions for the various test conditions are
shown in Figure 2. UVC achieved greater than 85% reduction in
virus aerosol concentrations for all test conditions. Increasing the
number of UVC fixtures from 1 to 4 resulted in greater fractional
reduction in virus aerosol concentration at both 2 and 6 ACH. The
fraction of virus surviving UVC treatment was lower under winter
conditions compared to summer conditions. The additional effective
air changes per hour due to UVC at each ventilation rate were 4 to
19 times greater during winter than summer.
Discussion
These data show that in a ‘real world’ test setup, upper-room
UVC is highly effective for reducing the concentration of vaccinia
virus aerosols. We demonstrate through aerosol decay experiments
that upper-room UVC fixtures used with mixing provided by a
conventional ceiling fan and minimal general ventilation produced
decreases in airborne virus concentrations that would require
additional ventilation of more than 87 ACH. During steady-state
experiments the combined effect of upper-room UVC and
ventilation had a nonlinear impact on the fraction of remaining
virus aerosol.[9] As a result, under winter conditions when
vaccinia is most susceptible to UVC inactivation, the effective
ACH due to upper-room UVC (ACH
UVC
) increased approxi-
mately five fold with increasing air exchange from ventilation. The
equivalent ventilation achieved by UVC ranged from a low of 18
to 1000 ACH
UVC
, with winter equivalent ventilation rates
consistently .100 ACH
UVC
.
The results from our decay experiments confirm the importance
of vertical mixing cited for UVC effectiveness in model
rooms.[3,10] Vertical mixing is required to move organisms from
the lower room into the upper-room where UVC intensity is the
highest. Without vertical mixing, some viruses may get less UVC
exposures or not get exposed at all and, as a result, UVC doses
may be insufficient to cause deactivation.[9] During our decay
experiments the ventilation system was operated so as to provide
UVC Inactivation of Poxviruses
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minimum negative pressure inside of the chamber to facilitate
aerosol containment, while providing minimal mixing and
dilution. As a result, when the heat boxes were not activated
vertical mixing was primarily attributable to convection associated
with the nebulizer diffuser which was heated to 37uC (approxi-
mately 17uC above the chamber temperature). The effective air
exchange rate was a modest 7 ACH
UVC
above the background.
The effective ACH rate more than doubled (16 ACH
UVC
) when
the heat given off by two people present in the room was simulated
by the activation of the heat boxes. This value is consistent with
the findings of Riley et el[11] for mycobacteria and are greater
than what would be achieved by recommended dilution
ventilation in hospital isolation rooms.[12] UVC and mixing
Figure 1. Background decay rates and decay rates for UVC light with and without heat boxes. a) ceiling fan is not operational; b) when
ceiling fan is operational.
doi:10.1371/journal.pone.0003186.g001
Table 1. Equivalent Air Changes per Hour Due to UVC
(ACH
UVC
) for Virus Aerosol Decay Tests with and without
ceiling fan and heat boxes.
Ceiling Fan Operational
No Yes
Without Heat Boxes 792
With Heat Boxes 16 87
doi:10.1371/journal.pone.0003186.t001
UVC Inactivation of Poxviruses
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using a ceiling fan together produced virus aerosol decay rates
equivalent to 87 ACH
UVC
and thus overwhelmed free convection
effects. Similarly, First et al were able to show marked reduction in
survival when comparing bacterial aerosol decay with and without
ceiling fans in operation.[9]
Although these data are strong indicators that UVC would be
an effective intervention, it has been recommended that tests of the
efficacy of UVC against bioaerosols be based on steady-state
measurements rather decay experiments.[10] We performed
steady-state experiments under both summer and winter condi-
tions. Consistent with early experiments on virus aerosol
stability[13,14], in the absence of UVC, vaccinia virus appeared
to be more stable and higher aerosol concentrations were achieved
with low RH (winter conditions) than with high RH (summer
conditions). These experimental results also show that upper-room
UVC is more effective when the relative humidity is low, even
though mixing was reduced by operating the ceiling fans on a low,
updraft, winter setting. These results are consistent with our
bench-top experiments showing that vaccinia aerosols are more
sensitive to UVC when relative humidity is low.[8]
Examining the fractional reduction of viral aerosol concentra-
tions under various conditions clearly shows that upper-room
UVC is capable of greatly reducing exposure. But, fraction
reduction measurements do not easily translate into estimates of
the actual level of risk achieved or facilitate decision making about
how to best deploy upper-room UVC as part of a protection
strategy. To estimate the level of risk with, for example, the Wells-
Riley equation, we need to convert the fractional survival
measurements into equivalent ventilation rates.[10,15] This is
easily done because at steady-state the ratio of total effective
sanitary ventilation (Q
UV
+Q) to actual ventilation through air
movement is equal to the ratio of virus concentration without
UVC to the concentration with UVC (the inverse of the remaining
fraction f
ss
, i.e. (Q
UV
+Q)/Q =f
ss
21
), where Q
UV
stands for the
supply of virus free air due to UV (see Appendix S1) and Q is the
ventilation rate with infective-virus-free air (m
3
/s).[9] If the
Table 2. Effective Air Changes per Hour Associated with Upper-Room UVC for Steady State Virus Aerosols.
Condition
Replicate Trials
(Air Samples)
Ventilation
Rate, ACH
UVC
Fixtures Fraction of Infective Virus Remaining ACH
UVC
Estimate
95% Confidence
Interval Estimate
95% Confidence
Interval
Summer 2 (20) 2 1 0.087 0.062 0.120 18 15 30
1 (12) 2 4 0.061 0.053 0.071 31 26 36
3 (36) 6 1 0.14 0.120 0.160 38 31 46
3 (36) 6 4 0.078 0.065 0.094 71 58 86
Winter 2 (24) 2 1 0.017 0.014 0.021 110 93 140
2 (24) 2 4 0.003 0.002 0.005 580 410 830
2 (24) 6 1 0.038 0.032 0.046 150 120 180
2 (24) 6 4 0.006 0.004 0.008 1000 740 1400
doi:10.1371/journal.pone.0003186.t002
Figure 2. Equivalent air changes due to UVC under steady state conditions with either 2 or 6 ACH, 1 or 4 UVC fixtures, or winter and
summer conditions.
doi:10.1371/journal.pone.0003186.g002
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fraction of infectious virus remaining were constant when the air
exchange was tripled, then the total effective sanitary ventilation
and effective ventilation due to UVC would also be tripled.
However, in these data, when we tripled the air exchange rate
from 2 to 6 ACH, the fraction of infectious virus remaining
increased. This does not, however, imply that upper-room UVC
gives less protection when ventilation is increased. It is true that
while increased ventilation reduced the virus aerosol concentration
it also reduced the average residence time of viral particles
resulting in lower UVC doses to individual particles. But, the
increase in fwas not great enough to completely offset the more
than additive effect of increased air exchanges. When we increased
the air exchange rate from 2 to 6 ACH, a factor of 3, the effective
ventilation due to UVC increased by a factor of 1.3 to 1.9. Thus,
increased ventilation actually increased UVC fixtures effectiveness
in terms of ACH
UV
– the combination of ventilation and upper-
room UVC is more than merely additive.
With one UVC fixture under summer conditions, when we
increased Qby 4 ACH the effective ventilation from UVC
increased by 20 ACH. In the winter with one fixture, when we
increased the air exchange by 4 ACH, the effective ventilation
from UVC increased by 40 ACH, and with 4 fixtures the effective
ventilation increased by 420 ACH. The high UVC susceptibility of
vaccinia when RH is low, i.e. the very small fobserved under
winter conditions, and the nonlinear interaction of UVC
disinfection with ventilation produced extremely highly effective
ventilation when the two were combined – ranging from .100 to
1000 ACH. In our previous bench top, dose-response studies of
vaccinia virus, moderate UVC doses (3 J/m
2
) reduced vaccinia
survival by a factor of .10,000 over natural biological decay.[8]
For the present study we used an equation developed by Rudnick
and First[16], that relates UVC fixture power output to mean
fluence rate for the entire room, to estimate the mean UVC dose for
the entire room assuming near perfect mixing, one fixture was in
use, and a ten minute exposure time (i.e. 6 AC/hr). Under these
conditions the UVC dose was estimated to be 17 J/m
2
. With four
fixures in use the dose would be expected to be 4-times higher.
Thus, the fraction of virus surviving, especially when they are most
susceptible, would be expected to be quite low.
Studies by other researchers have made similar measures of
UVC light effectiveness under steady-state conditions with
bacterial aerosols. Bacteria such as bacillus subtillus and serratia
marcescens have been used in full scale tests of upper-room
UVC[4,9] Bacteria are much more resistant to UVC light and
have a correspondingly lower UVC susceptibility parameter,
referred to as a Z-value. Riley and Kaufman noted decreased
susceptibility to UVC for Serratia Marcescens exposed to UVC when
RH exceeded 60% RH.[19] Ko et al noted a similar RH trends
with S. marcescens and Mycobacterium bovis aerosols exposed to
UVC.[20] Our previous studies of Vaccinia virus aerosol showed
vaccinia virus susceptibility was highest when relative humidity
was low.[8] Thus, the very high UVC susceptibility of vaccinia
virus, especially when relative humidity is low [8], most likely
accounts for the extremely high effective air changes per hour
associated with UVC we found in comparison to studies[3] using
bacterial aerosols. First et al. evaluated vaccinia virus in a full-scale
chamber under steady-state conditions at 50% RH and reported
similar results to our summer conditions.[9]
The additional effective ventilation due to upper-room UVC,
even in the summer, ranged between 18 and 71 ACH, rates in
excess of what is usually achieved in hospital rooms designed for
airborne precautions (approximately 12 ACH) [12]. The addi-
tional effective ventilation achieved under winter conditions was
phenomenal (110 with a single fixture to 1000 ACH with four
fixtures). These data demonstrate that upper-room UVC has the
potential to greatly reduce exposure to susceptible viral aerosols.
The greater survival at baseline and greater UVC susceptibility of
vaccinia under winter conditions suggest that while risk from an
aerosol attack with smallpox would be greatest in winter,
protective measures using UVC may also be most efficient at this
time. These data may also be relevant to influenza, which also has
improved aerosol survival at low RH. Given current concern
about potential for a pandemic in the near future, and the
potential that an important fraction of influenza transmission
occurs via aerosols, further studies of UVC susceptibility and
upper-room UVC effectiveness for influenza are warranted.
Materials and Methods
Experimental Chamber
The testing chamber, ante room, aerosol generation, and
sampling arrangements have been described previously and are
shown in Figure 3.[9] Briefly, virus aerosols were delivered at
1.5 meters above the floor in the center of a climate controlled
4.60 m62.97 m63.05 m high room equipped with a ceiling fan
and two black boxes containing 100-watt light bulbs (simulate
body heat of two people). The boxes were located approximately
one meter from the center of the room. UVC light was provided
by combinations of 5 wall-mounted Hygeaire UVC fixtures
(Model LIND24-EVO; Atlantic Ultraviolet Corp., Bay Shore,
NY), each using one 25-W low pressure mercury discharge lamp
with a UVC output of 5W. Fixtures were mounted 2.3 m from the
floor and experiments with one fixture used a single fixture pointed
down the middle of the long axis of the room, while experiments
with four fixtures used two fixtures on each end of the long axis
mounted one meter from the wall corners.
Aerosol generation and sampling
Vaccinia virus stock, Western Reserve strain, was prepared to a
concentration of 10
7
–10
8
plaque forming units (PFU)/ml as
reported previously[8]. Vaccinia stock solution was suspended in
phosphate buffered saline (PBS) with 10% fetal bovine serum and
20 ml of Antifoam A (Sigma, St. Louis). Vaccinia virus aerosols
were generated using a 6-jet Collison nebulizer (BGI Inc.,
Waltham, MA) operating at 138 kPa. The nebulizer was located
Figure 3. Schematic diagram of aerosol chamber and equip-
ment. Note: for clarity ceiling fan is not shown, but is located in the
center of the main chamber directly above the virus distributor. For
decay and single fixture steady state experiments the fixture shown in
the center of the wall on the left of the figure was used and for the four
fixture steady state experiments the other four fixtures were used.
doi:10.1371/journal.pone.0003186.g003
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in a class II biological safety cabinet (BSC) in the ante room and
attached to a permanently installed pipe leading to the center of
the test chamber. An omni-directional diffuser was attached to the
end of the pipe at 1.5 meters above the floor. The pipe was heated
to (37uC).
A port for aerosol sampling was located in front of the exhaust
grill and was connected via a pipe to a valve located within the
BSC in the control room. Air was drawn through a two-way valve
into either a 37 mm gelatin filter (SKC, Inc.Eighty Four, PA)
housed in a polyethylene cassette or through a bypass at 28.3 lpm.
Bypass or filtered air was then directed through a high efficiency
particulate air (HEPA) filter located before the high volume
sampling pump. The bypass was used to clear the dead space in
the sampling tube prior to sampling (60 sec) and when changing
the filters. Filters were dissolved and vaccinia viruses were
enumerated by plaque assay on confluent layers of Vero cells as
previously described.[8]
Decay experiments
Vaccinia was aerosolized for approximately 30 minutes to
achieve sufficiently high concentrations of virus to allow detection
after multiple logs of reduction. The generation was stopped and
5-minute samples were taken at 5 to 10 min intervals for up to
90 min. The aerosolization and sampling procedure was repeated
with one UVC fixture on (Figure 3) alternating with no UVC
fixture decay runs. Each experiment consisted of three pairs of
runs with UVC on and off. Decay experiments were carried out
without heat boxes or ceiling fan, with heat boxes, and with heat
boxes and the ceiling fan.
Steadystate
UVC inactivation of vaccinia virus was tested under steady state
conditions while simulating indoor summer (20uC, 80% RH,
ceiling fan directing air downwards) and indoor winter (20uC, 40%
RH, ceiling fan directing air upwards) environmental conditions,
with either 2 or 6 ACH ventilation rates, and either 1 or 4 UVC
light fixtures (Figure 3). We assumed that 3 air changes were
sufficient to establish a 95% chamber equilibration. Thus, virus
suspension was nebulized for 30 minutes prior to sampling to
achieve steady-state at 6 ACH and 1.5 hours for 2 ACH.
Triplicate sequential samples were collected with the UV fixtures
off followed by activation of the UVC fixtures and 3 air changes to
allow equilibration. Then, triplicate sequential samples were
collected with the UVC fixtures on. The fixtures were then
turned off and the cycle of sampling with fixtures off was repeated.
Each sample was assigned a time of collection as the midpoint of
the sampling interval relative to the start of virus nebulization.
Data Analysis
Decay experiment observations for the number of pfu/m
3
for
each sample were divide by the pfu/m
3
in the initial sample collected
after aerosolization was complete (collected approximately from t = 0
and t =5 min) to obtain an estimate of the fraction of infectious virus
remaining at each time point within an experiment. Each estimate of
fraction remaining was assigned to the midpoint of the sampling
interval. Thus, t = 2.5 min was assigned the preliminary value 1.0 for
fraction remaining. An exponential decay curve was fit to these data
using following equation:
fd~e{kt
where f
d
is the fraction of infectious virus surviving and kis a rate (or
decay) constant, and tis time. Each estimate of the fraction
remaining was then adjusted by dividing all fractions remaining by
the y-intercept of the regression. These adjusted fraction remaining
estimates from each of the triplicate experiments with the same
conditions were combined in a single regression to estimate the
exponential decay constant (i.e. the effective air exchange rate). The
equivalent air exchange rate due to UVC is the difference between
the decay constant when UVC is in use and when it is not.[9]
For steady-state experiments, we computed pfu/m
3
for each air
sample. For each set of experimental conditions, we regressed
ln(pfu/m
3
) on time, time squared, an indicator variable for
operation of the UVC fixtures, an indicator for each experiment,
and interactions of experiment indicators with the time variables.
This allowed us to determine the effect of UVC controlled for
experiment specific time trends in nebulizer output and variations
in the virus aerosol concentrations achieved in each replicate
experiment. The resulting coefficient for the indicator of UVC
operation was the log of f
ss
, the ratio of steady state concentration
of infectious virus with and without UVC, averaged over the
replicate experiments. The ACH due to UVC was then computed
as l
U
=l
o
(12f)/f
ss
where l
o
is the ACH due to ventilation (See
Appendix S1 for derivation). Regression analyses and confidence
limits for regression coefficients were computed using R statistical
software (R-Project, Version 2.6.0) and summarized in Excel
(Microsoft Corp, Redmond, WA).
Supporting Information
Appendix S1 Derivation of Equivalent Air Exchange Rate Due
to UVC. Derivation of equation used in data analysis.
Found at: doi:10.1371/journal.pone.0003186.s001 (0.03 MB
DOC)
Acknowledgments
The authors thank Kevin Banahan and Drew Rholl for their sampling and
analysis efforts during the aerosol experiments.
Author Contributions
Conceived and designed the experiments: JJM DKM SNR MWF.
Performed the experiments: JJM. Analyzed the data: JJM DKM SNR
MWF. Wrote the paper: JJM.
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