Aerosol Susceptibility of Influenza Virus to UV-C Light

Abstract

The person-to-person transmission of influenza virus, especially in the event of a pandemic caused by a highly virulent strain
of influenza, such as H5N1 avian influenza, is of great concern due to widespread mortality and morbidity. The consequences
of seasonal influenza are also substantial. Because airborne transmission appears to play a role in the spread of influenza,
public health interventions should focus on preventing or interrupting this process. Air disinfection via upper-room 254-nm
germicidal UV (UV-C) light in public buildings may be able to reduce influenza transmission via the airborne route. We characterized
the susceptibility of influenza A virus (H1N1, PR-8) aerosols to UV-C light using a benchtop chamber equipped with a UVC exposure
window. We evaluated virus susceptibility to UV-C doses ranging from 4 to 12 J/m2 at three relative humidity levels (25, 50, and 75%). Our data show that the Z values (susceptibility factors) were higher
(more susceptible) to UV-C than what has been reported previously. Furthermore, dose-response plots showed that influenza
virus susceptibility increases with decreasing relative humidity. This work provides an essential scientific basis for designing
and utilizing effective upper-room UV-C light installations for the prevention of the airborne transmission of influenza by
characterizing its susceptibility to UV-C.

Full text

Aerosol Susceptibility of Influenza Virus to UV-C Light
James J. McDevitt,
a
Stephen N. Rudnick,
a
and Lewis J. Radonovich
b
Harvard School of Public Health, Boston, Massachusetts, USA,
a
and National Center for Occupational Health and Infection Control, Veterans Health Administration,
Gainesville, Florida, USA
b
The person-to-person transmission of influenza virus, especially in the event of a pandemic caused by a highly virulent strain of
influenza, such as H5N1 avian influenza, is of great concern due to widespread mortality and morbidity. The consequences of
seasonal influenza are also substantial. Because airborne transmission appears to play a role in the spread of influenza, public
health interventions should focus on preventing or interrupting this process. Air disinfection via upper-room 254-nm germi-
cidal UV (UV-C) light in public buildings may be able to reduce influenza transmission via the airborne route. We characterized
the susceptibility of influenza A virus (H1N1, PR-8) aerosols to UV-C light using a benchtop chamber equipped with a UVC ex-
posure window. We evaluated virus susceptibility to UV-C doses ranging from 4 to 12 J/m
2
at three relative humidity levels (25,
50, and 75%). Our data show that the Z values (susceptibility factors) were higher (more susceptible) to UV-C than what has
been reported previously. Furthermore, dose-response plots showed that influenza virus susceptibility increases with decreasing
relative humidity. This work provides an essential scientific basis for designing and utilizing effective upper-room UV-C light
installations for the prevention of the airborne transmission of influenza by characterizing its susceptibility to UV-C.
Seasonal influenza is a major cause of morbidity and mortality
in the United States and throughout the world. Each year,
influenza accounts for about 3 million hospital days, 31 million
outpatient visits, and $10 billion in excess costs in the United
States. These consequences are most predominant in the very
young and the elderly (22). As demonstrated by the 2009 H1N1
influenza pandemic, influenza can spread rapidly in populations,
and the development of vaccines can take months to accomplish
(11). Furthermore, the use of nonpharmaceutical interventions
appears to have had limited effectiveness (11). In the event of the
pandemic spread of highly virulent strains of influenza, such as
H5N1 bird flu, which has recently shown a mortality rate of ap-
proximately 60%, effects could be disastrous (5, 26). For these
reasons, effective interventions to prevent the transmission of in-
fluenza are needed.
Contemporary science continues to study and understand the
modes used by influenza virus to spread from person to person.
There is debate about whether influenza virus is transmitted via
fine aerosols that are airborne, through exposure to large ballistic
droplets, or contact with fomites (3, 29). Understanding the route
of transmission is critical for implementing the best control strat-
egies. There is considerable evidence for the airborne transmission
of influenza via fine aerosols: influenza aerosols have been shown
to remain infective in laboratory experiments; the aerosol trans-
mission of influenza has also been shown to occur in animal and
human volunteer studies (1, 17); influenza nucleic acids have been
associated with fine particles in the exhaled breath of persons in-
fected with influenza virus (2, 7, 15, 16); and human epidemiology
studies have associated influenza transmission with airborne
routes (20, 23). As such, interventions to prevent influenza trans-
mission must go beyond the traditional dogma of cough etiquette,
hand hygiene, and social distancing. Due to their very low termi-
nal settling velocity, infectious fine particles, wafted by air cur-
rents, would be expected to remain airborne for hours. One inter-
vention proposed to be effective is UV irradiation, also called
UV-C light, emitted from specialized lamps placed near the ceiling.
The germicidal effect of light in the UV-C electromagnetic
spectrum (specifically 254-nm light) has been recognized for
some time. The susceptibility of microorganisms to UV-C light
varies and has been summarized in previous investigations (14).
Microorganism susceptibility to UV-C light is traditionally
thought to follow first-order kinetics according to the equation
F
R
⫽C
UV
/C
No UV
⫽e
⫺ZD
, where F
R
is the fraction remaining, C
UV
is microorganism concentration with UV exposure, C
No UV
is mi-
croorganism concentration without UV exposure, Zis the suscep-
tibility parameter expressed in m
2
/J, and Dis the dose of UV-C in
J/m
2
(4, 8, 18). These susceptibility parameters, or Z values, do not
appear to be static but vary with environmental conditions, such
as relative humidity (RH) (12, 13, 18). There is also evidence that
the susceptibility of microorganisms to UV-C light does not nec-
essarily follow first-order kinetics (18). Thus, it is necessary to
determine Z values in a defined and controlled experimental sys-
tem to predict UV-C effectiveness, determine the amount of
UV-C energy required for intervention, and develop models of
UV-C effectiveness.
Traditionally, UV-C light has been used to sanitize air using
UV-C light emitted by lamps located in the upper portions of
rooms (8). High-intensity light restricted to the upper part of the
room by special louvered fixtures inactivates microorganisms as
they circulate through the room via air currents. Due to the lou-
vered fixtures, the UV levels in the lower, occupied part of the
room remain at safe levels (25). The aim of our investigation was
to determine Z values for influenza virus aerosols at low, medium,
and high relative humidity.
MATERIALS AND METHODS
Virus preparation. A suspension of influenza virus (A/PR/8/34 H1N1),
which was purchased from Advanced Biotechnologies Inc. (Columbia,
Received 26 September 2011 Accepted 22 December 2011
Published ahead of print 6 January 2012
Address correspondence to James J. McDevitt, jmcdevit@hsph.harvard.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.06960-11
1666 aem.asm.org 0099-2240/12/$12.00 Applied and Environmental Microbiology p. 1666 –1669

MD), was thawed, divided into single-use portions, and stored at ⫺80°C
until needed.
Virus infectivity assay. A fluorescent focus reduction assay was used
to enumerate numbers of infective viruses and has been described previ-
ously (9, 28). Briefly, infected Madin-Darby canine kidney (MDCK) cells
(ATTC CCL-34) containing influenza A nucleoproteins were labeled with
influenza A virus nucleoprotein antibody (Abcam, Cambridge, MA) and
subsequently labeled with rhodamine-labeled goat anti-mouse IgG (Jack-
son ImmunoResearch Laboratories, West Grove, PA). The number of
cells having resulting fluorescent foci (measured in fluorescent focus units
[FFU]) was counted using an Olympus CKX-41 inverted fluorescence
microscope (200⫻total power; Olympus, Center Valley, PA). The num-
ber of FFU per sample was computed based on dilution factors and the
fraction of the well counted.
Benchtop UV-C aerosol exposure chamber. Influenza aerosols were
generated by adding 0.075 ml of undiluted influenza virus and 75 ml of
buffer (Dulbecco’s phosphate-buffered saline with calcium and magne-
sium containing 0.1% bovine serum albumin) into a high-output ex-
tended aerosol respiratory therapy (HEART) nebulizer (Westmed, Tuc-
son, AZ) and pressurizing at 69 kPa. The nebulizer output was mixed with
dry or humidified air (to achieve the desired RH level) in a 7.5-liter cham-
ber prior to delivery to the aerosol exposure chamber. The details of the
one-pass, dynamic aerosol test system were described previously (18). RH
and temperature in the chamber (measured via an Omega RH32 temper-
ature and relative humidity meter [Omega Engineering Inc., Stamford,
CT]) were measured prior to entry into the UV-C exposure section. UV-C
was generated by six 36-W low-pressure mercury lamps (254-nm light;
Lumalier, Memphis, TN), and screens were placed between the UV-C
light source and the exposure window to attenuate UV-C output and dose
within the exposure chamber. UV-C irradiance in the chamber was mea-
sured through a fused quartz port in the bottom of the UV-C exposure
section of the chamber using an IL 1400A UV light meter (International
Light, Peabody, MA). The irradiance levels used were based on initial
influenza virus dose-response experiments performed in our chamber
and the detection limits of our infectivity assay. The UV-C dose was com-
puted by multiplying the UV-C irradiance by the exposure time. The
exposure time was computed by dividing the volume of the chamber by
the airflow rate.
Air was drawn through the chamber by a pump at 25 liters/min
through a manifold attached to 2 SKC Biosamplers (SKC Inc., Eighty
Four, PA), each operating at 12.5 liters/min. Each Biosampler contained
20 ml of virus buffer (Dulbecco’s phosphate-buffered saline with calcium
and magnesium containing 0.1% bovine serum albumin). A HEPA filter
was connected after the samplers to remove fugitive aerosols before the
airstream entered the pump. When sampling was not in progress, the
aerosol-laden airstream running through the chamber was bypassed
around the samplers, and the 25-liter/min flow was directed to the HEPA
filter. The entire apparatus was set up inside a 6-foot class II biosafety
cabinet and maintained under negative pressure with respect to the cabi-
net interior.
The nebulizer was run for 20 min before sampling to ensure that
concentrations within the chamber had stabilized. Samples were collected
by passing the entire chamber airflow through the Biosamplers for a pe-
riod of 15 min. Sample pairs were collected that consisted of a sample with
the UV-C lights on followed by a control sample with the UV-C lights off.
Triplicate sample pairs were collected for combinations of UV-C dose
(ranging from 4 to 12 J/m
2
) and RH (25 to 27%, 50 to 54%, and 81 to
84%). After each sampling the BioSamplers were removed from the
chamber, the volume of collection liquid was measured, and virus collec-
tion fluid was stored at 4°C for a maximum of 3 h prior to performing the
infectivity assay. The BioSamplers were decontaminated with 10% bleach,
rinsed with 70% ethanol, and dried before reusing.
The fraction of virus surviving for each sample pair was calculated by
dividing the number of FFUs per sample with the UV-C lights on by the
number FFUs per sample with the UV-C lights off.
Linear regression through the origin was used to estimate the Z values
for each level of humidity. The ⫺log(F
R
) was our outcome variable, and
the dose level was our response. The estimated coefficient of the regression
line then was our estimate of the Z value. The bootstrap method was used
to determine whether the estimated Z values differed. Given any two Z
values that were tested, we bootstrapped 1,000 pairs of resampled data sets
from the original data at each humidity level, reran the regression models,
and calculated the difference between the estimated coefficients. This gave
us an estimate of the distribution of the difference between the coeffi-
cients. We calculated a Pvalue for each difference by using this distribu-
tion to test the hypothesis that the true difference is equal to 0. Statistical
analysis was performed using SAS 9.1 (SAS Institute, Cary, NC).
RESULTS
The average airborne concentration of influenza virus in the
chamber as measured by the focus assay was 5.62 ⫻10
3
FFU per
liter of air, which corresponded to 2,636 FFU per well on the
96-well plate. The fractional survival of influenza aerosols was
measured in triplicate for combinations of three ranges of relative
humidity (low, 25 to 27%; medium, 50 to 54%; and high, 81 to
84%) and 6 UV doses ranging from 4.9 to 15.0 J/m
2
(Fig. 1). Using
our experimental system, we measured influenza reductions as
low as 98.2% by comparing samples with the UV light on to sub-
sequent samples control samples with the UV light off. The coef-
ficients of variation between triplicate experiments ranged from
0.04 to 0.45 with a median of 0.13. The fractional survival of in-
fluenza aerosolized at low (25 to 27%), medium (50 to 54%), or
high (81 to 84%) RH is shown in Fig. 1. We calculated Z values of
0.29, 0.27, and 0.22 m
2
/J for low, medium, and high relative hu-
midity levels, respectively (Table 1). Generally, as the RH in-
creased the Z value decreased. The dose-response relationship ap-
pears linear when the log of the fractional survival is plotted
against the UV-C dose and reflects first-order decay. Linear re-
gression models fit the data well, as reflected by R
2
values, which
were at least 0.95. Bootstrap results of the 1,000 samples show that
all Z values are significantly different from each other (P⬍
0.0001).
DISCUSSION
The Z values determined for influenza aerosols in our study sug-
gest that influenza will be effectively inactivated during exposure
to upper-room UV-C. The Z value reported for influenza virus is
within the range of those reported for Mycobacterium tuberculosis.
Upper-room UV-C has been shown to be highly effective in lab-
oratory studies and has been proposed to control exposures to
tuberculosis (6, 12, 21, 24, 27). The influenza Z values were much
higher (i.e., influenza is more susceptible to UV-C) than those
reported for hardy spores, such as those from Bacillus anthracis
(Table 2) (4, 14).
The Z values reported herein all are higher than those reported
previously for influenza virus aerosols in a review by Kowalski et
al. (14), which was based on research by Jensen (10), and suggest
an increased efficacy of UV-C for deactivating influenza virus.
Jensen’s experiments were not designed to measure a Z value for
influenza, and in fact he did not calculate a Z value (18). However,
by making assumptions that were not explicitly supported by Jen-
sen’s description of his experiments, Kowalski was able to calcu-
late a Z value of 0.15 m
2
/J (18). The differences in Z values between
our data and previously reported data, although modest, may cor-
respond to large differences in virus survival. For example, for a
Influenza UV-C Light
March 2012 Volume 78 Number 6 aem.asm.org 1667

dose of 6 J/m
2
a Z value of 0.15 m2/J would result in a 60% reduc-
tion, while a Z value of 0.25 would result in a 77% reduction.
The Z values for influenza virus were statistically different as a
function of RH. These differences showing that influenza aerosols
are less susceptible to UV at higher RH than at lower RH have been
noted in previous UV susceptibility studies for other organisms
(18, 19). In climate-controlled, indoor environments, high RH
values near 75% may be unusual, but in areas without environ-
mental controls UV effectiveness may decrease in the presence of
elevated RH. In these instances where UV-C may be a cost-
effective intervention to reduce influenza exposure, more UV-C
energy may be required.
In recent studies of pox virus susceptibility to UV-C light, the
decay was not linear (18) but rather fit a model where a log-
normal distribution of susceptibilities described virus susceptibil-
ity. These differences may be due to the differences between pox
viruses and influenza virus. Pox viruses are large DNA viruses with
a high degree of structure, whereas influenza viruses are smaller,
simpler, RNA viruses. UV-C light may have different interactions
with nucleic acids to form photodimers, with thiamine found in
DNA and uracil found in RNA, and this may be the source of the
difference in dose-response.
The hospital environment is a location where the unchecked
transmission of influenza virus infection may lead to a hospital-
associated disease outbreak (26, 27). Despite increasing attention
to hospital infection control precautions in recent years, the ex-
posure of health care workers, patients, and visitors to influenza
and other respiratory infections remains a substantial problem
(28). Among the most formidable hospital-associated outbreaks is
one involving a highly virulent virus, such as the H5N1 virus cur-
rently circulating in Eurasia that is known to cause death in an
alarming fraction of its victims (29). To accommodate a surge in
patients needing health care during a severe pandemic, hospitals
are expected to house infected patients in groups consisting of
those with confirmed or suspected pandemic infection. In this
scenario, patients may be held in close proximity to each other on
a designated ward (section) of the facility. Limited numbers of
negative-pressure isolation rooms will be available, producing a
crowded environment with substantial airflow limitations.
Upper-room UV-C light could be utilized in this and many other
pandemic scenarios for air disinfection. Lindsley et al. and Bal-
chere et al. detected influenza virus nucleic acid associated with
fine particles in an emergency department and throughout a
health care facility (2, 15). Although the presence of nucleic acid
does not confirm the presence of infectious viruses, it is strong
evidence that viruses are being released into the air. The use of
upper-room UV-C light would provide cost-effective air disinfec-
tion in settings where it would not be feasible to use engineering
controls, such as increased ventilation rates or filtration (4). Fur-
thermore, the use of upper-room UV would not be limited to
controlling the spread of influenza virus, as it would also control
the spread of other airborne infectious agents (many of which are
susceptible to UV-C light) (14).
TABLE 2 Z values reported for various microorganisms
a
Microorganism Z value (m
2
/J)
Influenza A virus 0.15
Mycobacterium tuberculosis 0.10–0.48
Bacillus anthracis 0.031
a
Data are from Kowalski et al. (14).
FIG 1 UV-C susceptibilities of influenza virus aerosols at 25 to 27%, 50 to 54%, and 81 to 84% RH (each data point is the average from triplicate experimental
trials; error bars denote one standard deviation).
TABLE 1 Estimated Z values for influenza aerosols determined at low,
medium, and high relative humidity
RH range (%)
Estimated Z
value
95% confidence
interval
R
2
Lower Upper
25–27 0.29 0.27 0.31 0.985
50–54 0.27 0.26 0.31 0.991
81–84 0.22 0.21 0.23 0.992
McDevitt et al.
1668 aem.asm.org Applied and Environmental Microbiology

Relative humidity is one important variable which may influ-
ence the germicidal activity of UV-C light against viruses. Nebu-
lization method, sampling time, aerosol concentration, and dose
are other factors which may play an important role in the germi-
cidal activity of UV-C light. The characterization of these factors
was beyond the scope of our study but warrants future investiga-
tion.
Conclusions. By characterizing the susceptibility of influenza
virus to UV-C exposure, this work provides an essential scientific
basis for designing effective upper-room UV-C installations for
the prevention of influenza virus infection transmitted from per-
son to person via an airborne route. The Z values determined for
influenza virus were higher (i.e., greater susceptibility to UV-C)
than those previously reported. Additionally, UV-C effectiveness
was shown to decrease with increasing relative humidity.
ACKNOWLEDGMENTS
We thank Jinyize Wang for her work in the laboratory.
The opinions expressed in this article are those of the authors and do
not necessarily reflect the opinions or positions of the Department of
Veterans Affairs, the Office of Public Health, or the National Center for
Occupational Health and Infection Control.
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