EPA/600/R-10/127 |December 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Development of a Methodology
to Detect Viable Airborne Virus
Using Personal Aerosol Samplers
U.S. EPA, Office of Research and Development
National Homeland Security Research Center
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Development of a Methodology
to Detect Viable Airborne Virus
Using Personal Aerosol Samplers
GANG CAO, FRANCOISE M. BLACHERE,
WILLIAM G. LINDSLEY, JOHN D. NOTI,
AND DONALD H. BEEZHOLD
PREPARED FOR:
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HOMELAND SECURITY
RESEARCH CENTER
CINCINNATI, OH 45268
AFFILIATION AND ADDRESS:
ALLERGY AND CLINICAL IMMUNOLOGY
BRANCH
HEALTH EFFECTS LABORATORY DIVISION
CENTERS FOR DISEASE CONTROL
NATIONAL INSTITUTE FOR OCCUPATIONAL
SAFETY AND HEALTH
MORGANTOWN, WV 26505
INTERAGENCY AGREEMENT NUMBER
DW7592259701
PROJECT NUMBER TCAD 2.8
"The findings and conclusions in this report have not been formally disseminated by the
National Institute for Occupational Safety and Health and should not be construed to represent
any agency determination or policy. "
U.S. EPA, Office of Research and Development
National Homeland Security Research Center
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Disclaimer
U.S. Environmental Protection Agency (EPA), National Homeland Security Research Center and
the Centers for Disease Control and Prevention (CDC), National Institute for Occupational Safety
and Health (NIOSH). under IA #DW-75-92259701 (CDC IA# C110-001), collaborated in the
development of the analysis procedure described here.
This report has been peer and administratively reviewed and has been approved for publication as a
joint EPA and CDC/NIOSH. Note that approval docs not necessarily signify that the contents rellect
the views of the Agencies. CDC and EPA do not endorse the purchase or sale of any commercial
products or services.
Questions concerning this document or its application should be addressed to:
Don Bee/.ho Id
Allergy and Clinical Immunology Branch
Health Effects Laboratory Division
Centers for Disease Control
National Institute for Occupational Safety and Health
Morgantown, WV 26505
304-285-5963
Zec.l@cdc.gov
Erin Silvcstri
Project Officer
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati. OH 45268
513-569-7619
Sil.vest.ri .Erin@epa. gov
If you have difficulty accessing this PDF document, please contact Kathy Nickel
(Nickel.KathvrfD.eDa.gov') or Amelia McCall (McCaHAmeiia@epa.gov') for assistance.
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Foreword
Following the terrorist events of 2001, the U.S. Environmental Protection Agency's (EPA) mission
was expanded to account for critical needs related to homeland security. Presidential directives
identified decontamination follow ing a chemical, biological, and/or radiological attack as one of
EPA's primary responsibilities. To provide scientific and technical support for EPA's expanded
role, EPA's National Homeland Security Research Center (NHSRC) was established. The NHSRC
research program is focused on conducting research and delivering products that improve the
capability of the Agency to carry out its homeland security responsibilities.
As a part of its responsibility. NHSRC is charged with delivering detection techniques that will
enable the rapid characterization of threats, the identification of specific contaminants to protect
workers and the public, and the planning for recovery operations. A lot of effort and resources
have been allocated to the development of molecular assays and culture techniques applicable to
pathogens; however, initial sample collection and preparation technologies lag in development. To
bridge this critical gap. EPA collaborated with the Centers for Disease Control and Prevention's
(CDC) National Institute for Occupational Safety and Health (NIOSH). EPA and NIOSH worked
together to examine the ability of the NIOSH bioaerosol sampler to collect viable airborne viruses
and to devise techniques to preserve the viability of airborne viruses during and following after
collection. Results of this evaluation will help inform environmental remediation and recovery
activities.
This report summarizes the evaluation and the corresponding study results.
Gregory D. Sayles. Ph.D.. Acting Director
National Homeland Security Research Center
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Project Team
Contributions of the following individuals and organizations to the development of this document
arc acknowledged:
Centers for Disease Control and Prevention
Gang Cao
Francoisc M. Blachcrc
William G. Lindslcy
John D. Noti
Donald H. Bee/hold
Stephen Morse
Betsy Weirich
U.S. Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
Sanjiv Shah
Erin Silvcstri
Jacky Rosati (Technical Reviewer)
Sarah Perkins, Student Services Contractor (EP09C000212)
Region 5
Mark Durno (Technical Reviewer)
Region 10 Laboratory
Stephanie Harris (Technical Reviewer)
V
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vi
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Contents
Disclaimer iii
Foreword iv
Project Team v
List of Figures viii
List of Tables ix
List of Acronyms x
Executive Summary xi
1.0 Introduction 1
2.0 Materials and Methods 3
3.0 Results 5
4.0 Discussion 23
5.0 Conclusions 25
6.0 References 27
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List of Figures
Figure 1. NIOSH two-stage bioaerosol cyclone sampler. 6
Figure 2. Calm-air settling chamber for collecting aerosolized particles, 7
Figure 3. Effect of supplemented HBSS on the collection of viable and total viral particles 14
Figure 4. EITect of 2% agar coating on the collection of viable and total viral particles 15
Figure 5. EITect of 0.1% mucin coating on the collection of viable and total viral particles 16
Figure 6. EITect of elect ret filter coating on the collection of viable and total viral particles 18
Figure 7. EITect of storage time on the samples collected by NIOSH and SKC samplers 19
Figure 8. A typical si/c distribution of aerosols in the calm-air chamber. 20
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List of Tables
Tabic 1. Summary of Experiments Performed for the EPA Project 8
Table 2. Total Influenza Viral Particles Collected by NIOSH Samplers in a Calm-air Chamber 9
Table 3. Total Influenza Viral Particles Collected by SKC Samplers in a Calm-air Chamber 10
Table 4. Viable and Total Influenza Viral Particles in the Initial Viral Suspensions and in the
Samples Collected by NIOSH Samplers in a Calm-air Chamber 11
Table 5. Viable and Total Influenza Viral Particles in the Initial Viral Suspensions and in the
Samples Collected by SKC Samplers in a Calm-air Chamber 12
Table 6. Viable Influenza Viral Particles Collected by NIOSH Samplers in a Calm-air Chamber. .. 13
Table 7. Distribution of Collected Viable and Total Airborne Influenza Virus in the Stage 1,
Stage 2 and Backup Filter of the NIOSH Samplers (Average of all the NIOSH
Samplers Used in the Project)11 21
Table 8. Distribution of Collected Viable and Total Airborne Influenza Virus in the Stage 1,
Stage 2 and Backup Filter of the NIOSH Samplers (The Individual Experiments for
the Effect of Collection Times) 21
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List of Acronyms
APS
Aerodynamic particle si/cr
ATCC
American Type Culture Collection
BSA
Bovine serum albumin
CDC
Centers for Disease Control
cDNA
Complementary DNA
CEID50
Chicken embryo infectious dose endpoint 50%
DMEM
Dulbecco's modified Eagle's medium
EMEM
Eagle's modified essential medium
EPA
Environmental Protection Agency
Eq
Equation
HBSS
Hank's balanced salt solution
MDCK
Madin Darby canine kidney
NHSRC
National Homeland Security Research Center
NIOSH
National Institute for Occupational Safety and Health
PBS
Phosphate buffer saline
PFU
Plaque forming unit
PTFE
Polvtctrafluorocthvlcne
qPCR
Quantitative polymerase chain reaction
RCE
Response capability enhancement
RH
Relative humidity
SAM
Standardized Analytical Methods for Environmental Restoration Following
Homeland Security Events
tcid50
Tissue culture infectious dose endpoint 50%
TPCK
L-1 -tosy lamido-2-pheny lethy 1 chloromethyl ketone
TVP
Total viral particles
VPA
Viral plaque assay
VRA
Viral replication assay
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Executive Summary
A unique two-stage cyclone aerosol sampler that can separate bioaerosols into three size fractions was developed by
the National Institute for Occupational Safety and Health (NIOSH). The air sampler was tested for the ability to collect
viable airborne viruses from a calm air chamber loaded with a surrogate virus. Influenza A was used as the surrogate
virus due to the potential for emerging strains of influenza to create a pandemic. The sampler's efficiency at collecting
acrosoli/cd particles for 30 mill from a calm-air chamber is essentially the same as that from the SKC BioSamplei®
(SKC Inc.. Eighty Four. PA) that collects particles directly into a liquid media (1.2 X 104 total viral particles per
liter of air (TVP/L of air) versus 1.3 X 104 TVP/L of air. respectively). The efficiency of the NIOSH air sampler is
relatively constant over the collection times of 15, 30, and 60 minutes. The recovery rate for viable viral particles with
the NIOSH sampler is approximately 59% of the virus collected and. thus, it surpasses the reported viable recovery
rate of most commercial samplers with the exception of the SKC sampler. Under our experimental conditions, viable
infectious virus was collected in all three fractions of the NIOSH sampler. The highest number of viable infectious
virus was found in the 1-4 jun fraction (48-55%) and the <1 jim fraction (26-41%) while the smallest amount was
found in the >4 jun fraction (11-19%). A viral replication assay, which is based on a coupled tissue culture infectious
dose endpoint 50%/quantitative polymerase chain reaction (TCIDvl/qPCR) assay, was developed to amplify viral copy
number and to increase the sensitivity of the assay to detect viable virus. After normalization for differences in the
air-flow volumes collected by the NIOSH and SKC samplers, the NIOSH sampler collected a mean 2.1 X 10s TVP/L of
air and the SKC sampler collected a mean 6.4 X 10s TVP/L of air. Results from the viral replication assay verified that
the NIOSH sampler retains viable recovery similar to the efficiency obtained by the viral plaque assay. Collection of
viral particles onto test tube walls coated with mucin or agar, or into test tubes filled with a v arious amount of Hank's
balanced salt solution, did not further improve viability.
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xii
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1.0
Introduction
The U.S. Environmental Protection Agency (EPA)
has identified the detection of pathogenic airborne
microorganisms following a terrorist attack as a critical
component of an effective response. Detection of such
pathogens would require validated sampling techniques
that could be used by multiple laboratories following
a homeland security event. To meet this requirement,
EPAs National Homeland Security Research Center
(NHSRC). along with other EPA divisions and sister
agencies, published Standardized Analytical Methods
for Use During Homeland Security Events, Revision 1.0
(September 2004, EPA/600/R-04/126) and Revision 2.0
(September 2005, EPA/600/R-04/126B). It was rctitlcd
in 2007 and published as Standardized Analytical
Methods for Environmental Restoration Following
Homeland Security Events (SAM) in revision 3.0
(February 2007,EPA/600/R-07/015), 3.1 (November
2007, EPA/600/R-07/136), 4.0 (September 2008,
EPA/600/R-07/126D), and 5.0 (September 2009,
EPA/600/R-07/126E) which contain suggested assays for
use by laboratories tasked with performing confirmatory
analysis of environmental samples following a homeland
security event.
Concern regarding human exposure to bioaerosols laden
with toxic agents has led to the development of a variety
of air sampling devices. However, data addressing
the efficacy of current samplers to detect viable and
infectious airborne viruses is sparse and points to the
need of a more efficient sampler (1). A two-stage
cyclone bioaerosol sampler has recently been developed
by the National Institute for Occupational Safety and
Health (NIOSH) (2). The NIOSH sampler is unique in
that it size-fractionates bioaerosols and collects them in
disposable centrifuge tubes, facilitating direct processing
of samples. As air is drawn into an inlet at 3.5 L/niin.
the first stage of the NIOSH sampler, particles that arc
>4 nm arc collected into a 15 ml centrifuge tube. In the
second stage, 1 to 4 jim particles arc collected into a 1.5
ml microcentrifuge tube, and particles that are <1 jini are
collected onto a 37 nun polytctralluorocthylcnc (PTFE)
filter.
In previous studies employing a calm air chamber to
evaluate the performance of the NIOSH bioaerosol
cyclone personal sampler, a nebulized suspension of
the FluMist®vaccine (Mcdiiiiniunc, Gaithcrsburg, MD),
which contains live, attenuated influenza virus, (3) was
tested. Quantitative polymerase chain reaction (qPCR)
analysis results demonstrated that the sampler effectively
captured and separated viral-laden particles based on
their aerodynamic size. Similarly, while conducting a
field study during the February 2008 influenza season,
aerosol samples were successfully collected and sizc-
fractionatcd by both personal and stationary samplers
situated in the West Virginia University Hospital
Emergency Department (2). With qPCR analysis. 53%
of the detectable viral RNA was found in the rcspirablc
fraction of the aerosol. Rcspirablc particles are defined
as those small enough to be drawn down into the
alveolar region of the lungs (4). Collectively, these
studies suggest the potential for airborne transmission
of influenza. However, the viability and potential
infcctivity of the captured viral aerosols were not
addressed during cither study. Development of the
methodologies to assess viability and infcctivity would
directly address any dangers posed by virus-containing
particles and improve the utility of the NIOSH
bioaerosol sampler for the collection of airborne viruses.
Numerous reports have shown that the viability
of airborne viruses is dependent on the virus type,
environmental conditions, and on the methods of
collection and kindling of bioaerosol samples (5). The
survival of airborne influenza, for example, has been
shown to greatly depend on the relative humidity, as well
as on ambient air temperature and ultraviolet radiation
levels (6). The number of viable influenza, measles,
and mumps virus recovered from a bubbling sampler
increases when a virus maintenance fluid is used in
the sampler rather than distilled water (7). Airborne
bacteriophages have been shown to retain viability
longer after collection when they refrigerated rather than
stored at room temperature (8).
The objectives of this project were to examine the ability
of the NIOSH bioaerosol sampler to collect viable
airborne viruses and to devise techniques to preserve
the viability of airborne viruses during and following
collection. During experimentation, influenza A virus
was used as the surrogate virus due to the potential of
newly emerging strains to create a pandemic. In this
study, we showed that viable infectious virus were
present in all three fractions of collected particles. The
highest percentages of viable influenza virus were found
in the 1-4 urn fraction (48-55%) and the < 1 \im fraction
(26-41%) while the smallest percentages were found in
the >4 uni fraction (11-19%). Further, significantly more
total and more viable viral particles were collected in the
<1 \im range by increasing the air sampling time beyond
15 mill. Attempts to increase the viability of acrosoli/cd
viruses by collecting them onto test tube walls coated
1
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with mucin or agar, or into test tubes filled with various
amounts of Hank's balanced salt solution (HBSS). did
not further improve viability.
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2.0
Materials and Methods
Cell Culture: Madin Darby canine kidney (MDCK)
cells (CCL-34) were purchased from the American Type
Culture Collection (ATCC™, Manassas, VA). Cells were
propagated and maintained in 75-cm2flasks (Corning*
CellBind® Surface. Corning. NY). Growth medium
for MDCK cells consisted of Eagle's minimal essential
medium (EMEM, ATCC) supplemented with 10% fetal
bovine serum (Hyclone Laboratories. Inc. Logan. Utah),
0.4 units/ml penicillin (Invitrogen™. Carlsbad. CA),
and 0.4 ng/inl streptomycin (Invitrogen). Cells were
incubated at 35°C in a humidified 5% CO, incubator
until -90% confluent.
Wrws-Influenza A/WS/33 (H IN 1) virus (VR-825, Lot#:
58023547 and Lot#: 58772128) was purchased from
ATCC. For each experiment, one vial (1 ml) of the
virus stock was diluted in 30 ml of Hank's balanced salt
solution (HBSS) supplemented with 0.1% bovine serum
albumin (BSA), 100 units/ml penicillin, and 100 ug/ml
streptomycin (supplemented HBSS). The freshly made
virus suspension was placed on ice during the course of
the experiments.
Viral Plaque Assay (VPA): MDCK cells were
trypsini/cd. washed and plated at a density of 2.0 x 106
per well (CoStar 6-well tissue culture plate. Corning).
Cells were incubated at 35°C in a humidified 5% CO,
incubator overnight. Confluent cellular monolayers
were next washed two times with phosphate buffered
saline (PBS, Invitrogen) and treated with serial dilutions
of the viral aerosol samples. Following 45 mill of
adsorption, influenza A/WS/33-infected MDCK cells
were washed with PBS, overlaid with an agarose
medium solution and incubated at 35°C in a humidified
5% CO, incubator for 48 h. Plaques were visually
enumerated and plaque forming units (PFU )/ml were
calculated.
Bioaerosol Samplers: A two-stage cyclone bioaerosol
sampler developed by NIOSH was used to collect
influenza virus-containing aerosols generated in the
laboratory. The NIOSH sampler consists of a 15 ml
centrifuge tube as the 1st stage, a 1.5 ml centrifuge tube
as the 2nd stage, and a backup polytctralluoracthylcnc
(PTFE filter). As a comparison, the SKC sampler (SKC
BioSampler® SKC Inc. Eighty Four. PA) was also used
for each experiment. The SKC sampler contained 15 ml
of supplemented HBSS in the 20 ml collection vessel.
Calm-Air chamber Aerosolization and Collection of
Influenza Virus: Diluted influenza virus suspension was
acrosoli/cd by a 1 -jet Collison nebulizer (BGI. Waltham,
MA) for the experiments (EPA01-EPA04) and by an
AeroNeb* ncbuli/cr (Aerogen*. Gahvay. Ireland) for the
experiments (EPA05-EPA19). The generated aerosols
were mixed in a mixing chamber with 30 L/min of air at
20% relative humidity (RH) through a dispersion nozzle
in the top center of a 40 L calm-air chamber. The forces
of gravity and inertia caused the aerosols to settle into
the bottom of the chamber where the NIOSH sampler
(i.e., replicates s and a sample inlet for the SKC samplers
(i.e., replicates) were located.
The chamber air was drawn by a Model 3321
Aerodynamic Particle Sizer®(TSI®, Shoreview. MN) at
5 L/min through a vertical probe at the same height as
the sampler inlets to monitor the aerosol concentration
and si/c distribution. The aerosols were collected by
both NIOSH and SKC samplers. The NIOSH samplers
were connected to personal air sampling pumps (Model
224-PCXR4; SKC, Eighty Four, PA, USA) and the
SKC samplers were connected to a central vacuum line.
The NIOSH samplers were positioned inside and at
the bottom of the calm air chamber, whereas, the SKC
samplers were placed outside the chamber with the
sampler inlet housed inside the chamber. Generally, for
each EPA experiment, multiple samplers were placed
inside and outside the chamber.
To collect the airborne influenza virus, after operation of
the AeroNeb ncbuli/cr for 10 ruin and once the aerosol
concentration in the chamber stabilized, the vacuum
pumps for the NIOSH samplers and the vacuum line for
the SKC sampler were switched on, simultaneously. The
ncbuli/cr provided continuous loading of aerosols in the
chamber. The NIOSH samplers collected the aerosols for
15. 30, and 60 mins at 3.5 L/min while the SKC sampler
collected the aerosols for 15 mins at 12.5 L/min. After
collection, the ncbuli/cr. the pumps and the vacuum
line were turned off. The exterior of the samplers were
wiped off with a clean, low lint, laboratory tissue to
remove the deposited particles. The NIOSH samplers
were disassembled for analysis.
The concentrations of aerosols loaded in the calm-air
chamber and the viable and total virus particles in the
initial viral suspensions varied among experiments,
which introduced artificial variations in concentrations
of viable and total influenza particles collected by the
samplers. To reduce such variations, we normalized
the concentrations [cone] of viable and total influenza
particles using Equation 1 and Equation (Eq) 2:
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normalized infectious cone (PFU/L of air) by the sampler i =
infectious cone (PFU/L of air) by the sampler i
total aerosols collected by the sampler i
V
average total aerosols collected
viable particles in the initial viral suspension
average viable particles in the initial viral suspension
Eq 1
normalized total virus cone (TVP/L of air) by the sampler i =
total virus conc(TVP/L of air) by the sampler i
total aerosols collected by the sampler i
V
average total aerosols collected
total viral particles in the initial viral suspension
average total viral particles in the initial viral suspension
Eq 2
The viability of virus was calculated by dividing the
viable influenza viral particles obtained from the VPA
by the total viral particles (TVP) per liter of air from
quantitative polymerase chain reaction (qPCR).
Viral RNA Isolation and cDNA (complementary
DNA) Transcription. Viral RNA was isolated from
aerosol samples using the MagMax™-96 Viral RNA
Isolation Kit ( Applied B iosy ste inss / A mb io 11 \ Austin,
TX). Briefly, following collection, aerosol samples
were suspended in 500 jj.1 of Lysis/Binding Concentrate
(Ambion) and stored at -20°C. Upon thawing. 500 jj.1 of
isopropanol was added to each sample to complete the
Lysis/Binding Solution preparation and viral RNA was
extracted according to the manufacturer's instructions.
The final eluted total-RNA volume was 32 jj.1. RNA was
immediately transcribed into cDNA using High Capacity
RNA to cDNA Master Mix ( Applied Biosysteins. Foster
City, CA). The final cDNA volume was 40 jj.1.
Real-Time qPCR Analysis. To detect influenza
virus, real-time qPCR analysis was performed using
the following matrix 1 gene primers (corresponding
to the 33 amino acids at the n-terminus as
described in Spackman et al. (9)): Forward 5'-
AGATGAGTCTTCTAACCGAGGTCG-3". Reverse 5'-
T G C A A A A A C AT CTT C A A G T CT CT G - 3' and probe:
6FAM-TCAGGCCCCCTCAAA GCC-MGBNFQ.
All primers and probes were synthesized by Applied
Biosysteins and used at a final concentration of 0.8
jiM and 0.2 jiM. respectively. The qPCR (45 cycles)
was performed with the Applied Biosysteins 7500 Fast
Real-Time PCR System as follows: 20 sec at 95 C
(initial denaturation), 3 sec at 95 C (amplification), and
30 sec at 60°C (extension). To determine the relative
viral genome copy, a standard curve was generated from
10-fold serial dilutions of the influenza Ml matrix gene
and analyzed concurrently with all qPCR reactions. A
negative control without template was also included in
all real-time PCR reactions. All reactions were run in
duplicate and averaged.
Viral Replication Assay (VRA): To assess viral
replication, a modified tissue culture infectious dose
endpoint 50% (TCID50) assay (Michael Shaw, PhD1,
personnel communication) was performed. Prior to viral
treatment. MDCK cells were trypsini/ed. washed and
re-suspended in (Dulbecco's modified Eagle's medium
(DMEM, Invitrogen) supplemented with 1 % bovine
serum albumin (BSA, Invitrogen), 25 mM HEPES
(Invitrogen). 2 ng/ml L-10 tosylainido-2-phenylet hy 1
chloromethyl ketone (TPCK) Trypsin (Sigma. St. Louis,
MO), 0.2 units/ml penicillin (Invitrogen), and 0.2 jj.g/
ml streptomycin (Invitrogen). Cells were plated in
quintuplicate at a density of 5.0 x 104 per well (CoStai®
flat bottom 96-well plate. Corning) and incubated
overnight at 35°C. Plated cells were next treated with
five serial dilutions of each viral aerosol sample at 35"C-
in a humidified 5% CO, incubator for 24 h. Following
treatment, cell culture supernatants/viral inoculums were
removed, cellular monolayers were washed with PBS
and cells were lysed in 66 jj.1 Lysis/Binding Solution
Concentrate (Ambion) and stored at -20°C until RNA
extraction.
VRA RNA Isolation and cDNA Transcription. Total
RNA was isolated from A/WS/33-infected MDCK
cells using the MagMax™-96 Total RNA Isolation Kit
(Ambion). Briefly, upon thawing of the lysed cellular
solution. 66 jj.1 of isopropanol was added to each sample
well to complete the Lysis/Binding Solution preparation
and total RNA was extracted according to the
manufacturer's instnictions. The final eluted total RNA
volume was 32 jj.1. RNA was immediately transcribed
into cDNA using High Capacity RNA to cDNA Master
Mix ( Applied Biosysteins). The final cDN A volume was
40 nl.
1 Michael W. Shaw, PhD, Influenza Division, MS G-16, Centers for Disease Control and Prevention, Atlanta, GA 30333, email: mshawl@cdc.gov
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3.0
Results
Recovery Efficiency of Viral Particles from the
NIOSH Sampler: The NIOSH sampler size-fractionates
bioaerosols and collects them in disposable collection
tubes or on a backup PTFE filter (Figure 1). Experiments
were conducted to determine the recovery efficiencies
of virus deposited directly onto the wall of a collection
tube or the backup filter. One hundred microliters of
a stock suspension of influenza A/WS/33 (5.27 X 106
virus/ml. ATCC VR-825) was spotted onto replicate
backup filters in 10 jj.1 aliquots. The spiked filters were
then dried under clean air at a flow rate of 3.5 L/iiiin for
0, 15, 30, or 60 min. The virus was then extracted from
each filter and the total number of virus recovered was
determined by the qPCR. As control, 100 jj.1 of stock
suspension was analyzed by the qPCR. The results (data
not shown) revealed that 17.1%, 10.3%, 10.8%, and
11.7% (mean of three replicates under each experimental
condition) of the spiked virus was recoverable from
filters dried for 0, 15, 30, and 60 min, respectively. The
VPA was used to assess the viable virus recovery and
the results showed that 60.9%, 57.3%, 52.5%, and 59.5
% (mean of three replicates under each experimental
condition) of the virus remained viable after 0, 15, 30,
and 60 min of drying before extraction. The recovery
of virus from multiple Stage 1 collection tubes was
similarly addressed. The recovery of total viral particles
was 15.7% and 15.9% from Stage 1 tubes that were
extracted immediately or dried under clean air for 30
min. respectively. The viable virus recovery was 65.7%
and 50.3% from the Stage 1 tubes that were extracted
immediately or dried under the clean air for 30 min,
respectively.
Viability Assessment of Collected Viral Particles:
A total of 19 calm-air chamber experiments (Figure 2)
were performed at room temperature with 20% humidity
for this project (Table 1). The objectives of these
experiments were to examine the ability of the NIOSH
sampler to collect viable airborne viruses and to test
the effects of the collection media, collection time and
sample storage time on viability of collected airborne
influenza virus.
In the early stage of this project (EPA01-EPA04,
unpublished data), a 1 -jet collision nebulizer was
used to aerosolize the influenza virus. The results,
however, showed a significant loss in viability with
this ncbuli/cr. We then tested whether the AeroNeb
ncbuli/cr would better preserve viability. The AeroNeb
ncbuli/cr was positioned directly on the opening of
a stage 1 collection tube and droplets coming out
from the bottom of the ncbuli/cr were collected and
used for the qPCR and VPA analyses. Samples were
collected on ice or at room temperature to determine
whether viability could be improved by lowering the
collection temperature. As control, a sample of the
viral suspension before nebulization was assayed. The
ncbuli/cr remained on for 30 min per each collection to
mimic the calm-air chamber experimental conditions.
The results showed tliat 42% of the total viral particles
acrosoli/cd were recovered in the collection tube placed
on ice and essentially no loss in viability was shown.
Similarly. 49% of the total viral particles acrosoli/cd
were recovered in the tube placed at room temperature
and viability did not decrease. These results show that
the AeroNeb ncbuli/cr docs not reduce viral viability.
Experiments using the calm-air chamber for collection
of acrosoli/cd virus were previously done at room
temperature, and. as such, t lie re is no advantage to
performing future experiments at colder temperatures.
All of the subsequent experiments described here
employed an AeroNeb ncbuli/cr.
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2nd stage
(1.5 ml
centrifuge
tube)
\ /
1st stage (15
ml
centrifuge
tube)
Figure 1. NIOSH two-stage bioaerosol cyclone sampler.
6
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AeroNeb
micropump
Mixing chamber nebulizer
Excess
aerosol
Aerosolized virus
Conditioned air
Calm-air
settling
chamber
Dry
air
Temperature
& humidity
monitor
NIOSH sampler
Humidifier
To aerosol
particle
counter
SKC BioSampler
Figure 2. Calm-air settling chamber for collecting aerosolized particles.
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Table 1. Summary of Experiments Performed for the EPA Project
Exp#
Nebulizer
Collec.
time
(min)
#of
NIOSH
samplers
Viability
detection
Note1
EPA-01
Collison
1-jet
60
6
VPA
Pilot for detection of viable airborne influenza virus
EPA-02
Collison
1-jet
60
2
VPA
Repeat EPA-01 but with a greater number of aerosolized
influenza virus loaded in the calm-air chamber
EPA-03
Collison
1-jet
60
3
VPA
Test the effect of sample storage time on viability of
influenza virus collected
EPA-04
Collison
1-jet
60
3
VPA
Test the effect of liquid medium (HBSS) on viability of
influenza virus collected
EPA-05
AeroNeb
30
3
VPA
Test a different nebulizer to generate airborne influenza
virus
EPA-06
AeroNeb
60
3
VPA
EPA-07
AeroNeb
30
3
VPA
Stage 1 of NIOSH samplers (Tl) was agar-coated
EPA-08
AeroNeb
60; 30
5
VPA
Test the collection time on the performance of NIOSH
samplers
EPA-09
AeroNeb
60; 30
4
VPA
EPA-10
AeroNeb
30; 15
4
VPA
EPA-11
AeroNeb
20; 30;
40; 50;
60
5
Luciferase
Test the luciferase method for detecting and quantifying
airborne influenza virus
EPA-12
AeroNeb
40
3
Luciferase;
VPA
EPA-13
AeroNeb
40
3
Luciferase;
VPA
EPA-14
AeroNeb
30
4
VPA
Test the effect of sample storage time on viability of
influenza virus
EPA-15
AeroNeb
30
4
VPA
Stage 1 ofNIOSH samplers (T1) was coated with 0.1%
mucin solution
EPA-16
AeroNeb
30
4
VPA
Test the new stock VR-825
EPA-17
AeroNeb
30
2
VRA
Test the viral replication assay for detecting and quantifying
airborne influenza virus
EPA-18
AeroNeb
30
2
VRA
Stage 1 and Stage 2 ofNIOSH samplers (Tl, T2) were
coated with electret filter
EPA-19
AeroNeb
30
5
VPA
Test the effect of liquid medium (HBSS) on viability of
influenza virus collected
Acronyms: EXPP, Experiment; HBSS, Hank's balanced salt solution; VPA, Viral plaque assay
'qPCR was performed to determine the total viral particles for all experiments except EPA-01 and EPA-02.
Stock VR-825 (Lot#: 58023547) was used for EPA-01-EPA15. Stock VR-825 (Lot#: 58772128) was used for EPA-16 through
EPA-19.
8
-------
We previously demonstrated that the collection of
FluMist®vaccine (Medimmune, LLC.; Gaithersburg.
MD) with the NIOSH two-stage sampler was linear at
least up to 40 min of sampling time (3). In that study,
however, the viability of the collected virus particles
was not assessed. To determine whether the NIOSH
sampler can collect viable virus, acrosoli/cd influenza A/
WS/33 was loaded into a calm air chamber equilibrated
to 21°C and 20%relative humidity, and air samples
were collected for 15, 30, and 60 in in in nine separate
experiments. For each experiment, 1 to 4 samplers were
simultaneously connected to the calm air chamber and
the results are shown in Table 2. Over the collection
periods of 15, 30, and 60 min, the TVP collected
increased linearly (averages of 2.53 X 106, 4.4 X 106,
and 8.74 X 106, respectively).
The total number of viral particles, pooled from the
collection tubes and filter, varied with the starting virus
concentration in the initial viral suspension (i.e., prior to
aerosoli/ation). However, within a given experiment.
the number of viral particles collected also increased
linearly. For example, in experiment number EPA-
10, the average total viral particles collected was 6.41
X 105 at 15 min and 1.04 X 106 at 30 min. Similarly,
in experiment number EPA-8, the average total viral
particles collected was 3.97 X 105 at 30 min and 8.93 X
105 at 60 min. Normalization of the number of collected
total virus to the amount loaded into the calm-air
chamber, the air flow rate, and the amount of time for
collection was performed to determine the efficiency of
the NIOSH sampler over the sampling time and also to
directly compare its efficiency with the SKC sampler.
The results revealed that the NIOSH sampler retained
similar collection efficiencies over the 15-60 min
sampling period in that 1.32 X 104 total viral particles
per liter (TVP/L) of air were collected after 15 in in of
sampling. 1.2 X 104 TVP/L were collected after 30 min.
and 2.68 X 104 TVP/L were collected after 60 min (Table
2). Moreover, the efficiency was essentially the same as
that obtained from the SKC sampler (Table 3, average
1.3 X 104 TVP/L).
Table 2. Total Influenza Viral Particles Collected by NIOSH Samplers in a Calm-air Chamber
Exp. #
Collect
Total aerosols
Total viral
Total viral
Total virus
Normalized total virus
time (min)
collected
particles (in initial
particles (TVP)
conc. (TVP/L
conc. (TVP/L of air)
viral suspension)
collected
of air)
collected
EPA-10
15
2.53E+09
1.03E+09
7.18E+05
1.37E+04
1.48E+04
EPA-10
15
2.53E+09
1.03E+09
5.63E+05
1.07E+04
1.16E+04
Average (SD)
1.32E+04 (2.26E+03)
EPA-05
30
4.07E+09
2.86E+08
6.20E+05
5.90E+03
2.30E+04
EPA-07
30
4.01E+09
1.94E+08
3.47E+05
3.30E+03
1.93E+04
EPA-08
30
4.85E+09
1.61E+08
3.97E+05
3.78E+03
2.21E+04
EPA-09
30
5.01E+09
3.88E+08
2.89E+05
2.75E+03
6.43E+03
EPA-09
30
5.01E+09
3.88E+08
1.79E+05
1.70E+03
3.99E+03
EPA-10
30
3.82E+09
1.03E+09
9.44E+05
8.99E+03
1.04E+04
EPA-10
30
3.82E+09
1.03E+09
1.13E+06
1.08E+04
1.25E+04
EPA-14
30
4.98E+09
2.10E+09
1.24E+06
1.18E+04
5.11E+03
EPA-14
30
4.98E+09
2.10E+09
2.60E+06
2.48E+04
1.08E+04
EPA-15
30
3.99E+09
3.61E+09
4.87E+06
4.64E+04
1.46E+04
EPA-15
30
3.99E+09
3.61E+09
4.82E+06
4.59E+04
1.45E+04
EPA-16
30
4.72E+09
1.31E+09
1.01E+06
9.66E+03
7.10E+03
EPA-16
30
4.72E+09
1.31E+09
1.27E+06
1.21E+04
8.88E+03
EPA-16
30
4.72E+09
1.31E+09
1.37E+06
1.31E+04
9.60E+03
EPA-16
30
4.72E+09
1.31E+09
9.30E+05
8.86E+03
6.51E+03
EPA-19
30
2.96E+09
8.32E+08
9.86E+05
9.39E+03
1.74E+04
Average (SD)
1.20E+04 (5.97E+03)
EPA-06
60
7.83E+09
3.36E+08
3.07E+06
1.46E+04
5.40E+04
EPA-08
60
8.96E+09
1.61E+08
8.85E+05
4.21E+03
2.84E+04
EPA-08
60
8.96E+09
1.61E+08
8.76E+05
4.17E+03
2.81E+04
EPA-08
60
8.96E+09
1.61E+08
9.20E+05
4.38E+03
2.95E+04
EPA-09
60
8.85E+09
3.88E+08
8.59E+05
4.09E+03
1.16E+04
EPA-09
60
8.85E+09
3.88E+08
6.88E+05
3.27E+03
9.26E+03
Average (SD)
2.68E+04 (1.61E+04)
Acronyms: TVP, Total viral particles
9
-------
To determine whether the viability of acrosoli/cd
virus is maintained following collection with the
NIOSH sampler, the percentage of viable virus in the
initial viral suspension was compared with that in the
collected sample (Table 4). In general, as determined
by a viral plaque-forming assay (VPA), the average
loss of viability was 41% (59% of the recovered virus
were viable). In contrast, there was no loss in viability
detected with the SKC sampler (Table 5). Prolonged
collection times have been shown to result in decreased
viral recovery (10). Therefore, to determine whether
the NIOSH sampler is able to collect viable virus
over an extended sampling period, collected samples
were assayed for infective virus using the VPA. As
shown in Table 6, the number of viable virus particles
collected per liter of air varied with each experiment as
a result of variability in the number of viable virus in
the initial suspension prior to aerosolization. However,
after normalization to correct for these differences, the
average PFU/L of air was 14.91 from samples collected
for 15 mill. 14.26 for samples collected for 30 mill. and
11.59 from samples collected for 60 mill. The variability
among the experiments within each collection period
was not statistically significant and more importantly,
there was essentially no adverse effect on viability with
prolonged collection of up to 60 niin.
Table 3. Total Influenza Viral Particles Collected by SKC Samplers in a Calm-air Chamber
Exp.#
Collect
time
Total aerosols
collected
Total viral
particles (in initial
viral suspension)
Total viral
particles (TVP)
collected
Total virus
conc. (TVP/L
of air)
Normalized total virus
conc. (TVP/L of air)
collected
EPA-05
15
7.76E+09
2.86E+08
9.25E+05
4.93E+03
1.76E+04
EPA-06
15
6.43E+09
3.36E+08
4.60E+05
2.45E+03
8.98E+03
EPA-07
15
6.54E+09
1.94E+08
7.89E+05
4.21E+03
2.62E+04
EPA-08
15
7.14E+09
1.61E+08
1.18E+06
6.31E+03
4.35E+04
EPA-08
15
7.33E+09
1.61E+08
1.01E+06
5.36E+03
3.60E+04
EPA-09
15
7.99E+09
3.88E+08
7.36E+04
3.93E+02
1.00E+03
EPA-09
15
5.90E+09
3.88E+08
4.48E+05
2.39E+03
8.27E+03
EPA-10
15
9.07E+09
1.03E+09
2.85E+06
1.52E+04
1.29E+04
EPA-10
15
3.95E+09
1.03E+09
1.73E+06
9.20E+03
1.80E+04
EPA-13
15
4.84E+09
6.46E+08
4.11E+05
2.19E+03
5.55E+03
EPA-14
15
8.99E+09
2.10E+09
1.23E+06
6.58E+03
2.75E+03
EPA-14
15
8.58E+09
2.10E+09
3.70E+06
1.97E+04
8.64E+03
EPA-15
15
6.19E+09
3.61E+09
9.59E+06
5.11E+04
1.81E+04
EPA-15
15
8.08E+09
3.61E+09
3.26E+06
1.74E+04
4.72E+03
EPA-16
15
8.14E+09
1.31E+09
1.60E+06
8.52E+03
6.32E+03
EPA-16
15
8.59E+09
1.31E+09
9.21E+05
4.91E+03
3.45E+03
EPA-19
15
5.23E+09
8.32E+08
7.32E+05
3.91E+03
7.11E+03
EPA-19
15
5.35E+09
8.32E+08
4.54E+05
2.42E+03
4.31E+03
Average (SD)
1.30E+04 (1.18E+04)
10
-------
Table 4. Viable and Total Influenza Viral Particles in the Initial Viral Suspensions and in the Samples Collected
by NIOSH Samplers in a Calm-air Chamber
Exp.#
Collect
Plaque
Total viral
Infectious to
Infectious
Total virus conc.
Infectious
time
forming
particles (TVP)
total (%) (in
conc. (PFU/L
(TVP/L of air)
to total (%)
(min)
units(PFU) in
initial viral
suspension)
(in initial viral
suspension)
initial viral
suspension)
of air)
(in collected
sample)
EPA-10
15
3.90E+06
1.03E+09
0.38
26.90
1.37E+04
0.20
EPA-10
15
3.90E+06
1.03E+09
0.38
28.10
1.07E+04
0.26
EPA-05
30
3.28E+06
2.86E+08
1.15
15.25
5.90E+03
0.26
EPA-07
30
1.61E+06
1.94E+08
0.83
14.96
3.30E+03
0.45
EPA-08
30
1.95E+06
1.61E+08
1.21
15.64
3.78E+03
0.41
EPA-09
30
2.10E+06
3.88E+08
0.54
10.87
2.75E+03
0.40
EPA-09
30
2.10E+06
3.88E+08
0.54
8.33
1.70E+03
0.49
EPA-10
30
3.90E+06
1.03E+09
0.38
25.83
8.99E+03
0.29
EPA-10
30
3.90E+06
1.03E+09
0.38
30.00
1.08E+04
0.28
EPA-14
30
1.91E+06
2.10E+09
0.09
14.64
1.18E+04
0.12
EPA-14
30
1.91E+06
2.10E+09
0.09
19.64
2.48E+04
0.08
EPA-15
30
2.59E+06
3.61E+09
0.07
15.12
4.64E+04
0.03
EPA-15
30
2.59E+06
3.61E+09
0.07
15.12
4.59E+04
0.03
EPA-16
30
7.88E+05
1.31E+09
0.06
5.63
9.66E+03
0.06
EPA-16
30
7.88E+05
1.31E+09
0.06
4.92
1.21E+04
0.04
EPA-16
30
7.88E+05
1.31E+09
0.06
4.88
1.31E+04
0.04
EPA-16
30
7.88E+05
1.31E+09
0.06
6.48
8.86E+03
0.07
EPA-19
30
1.13E+06
8.32E+08
0.14
10.83
9.39E+03
0.12
EPA-06
60
4.80E+06
3.36E+08
1.43
35.35
1.46E+04
0.24
EPA-08
60
1.95E+06
1.61E+08
1.21
12.37
4.21E+03
0.29
EPA-08
60
1.95E+06
1.61E+08
1.21
11.19
4.17E+03
0.27
EPA-08
60
1.95E+06
1.61E+08
1.21
10.65
4.38E+03
0.24
EPA-09
60
2.10E+06
3.88E+08
0.54
8.37
4.09E+03
0.20
EPA-09
60
2.10E+06
3.88E+08
0.54
7.74
3.27E+03
0.24
-------
Table 5. Viable and Total Influenza Viral Particles in the Initial Viral Suspensions and in the Samples Collected
by SKC Samplers in a Calm-air Chamber
Exp.#
Collect
time
(min)
PFU (in
initial viral
suspension
Total viral
particles (in
initial viral
suspension)
Infectious to
total (%) (in
initial viral
suspension)
Infectious
conc.
(PFU/Lof
air)
Total virus
conc. (TVP/L
of air)
Infectious
to total (%)
(in collected
sample)
EPA-05
15
2.93E+06
2.86E+08
1.02
69.33
4.93E+03
1.41
EPA-06
15
2.40E+06
3.36E+08
0.71
76.27
2.45E+03
3.11
EPA-07
15
1.39E+06
1.94E+08
0.72
38.03
4.21E+03
0.90
EPA-08
15
1.95E+06
1.61E+08
1.21
90.00
6.31E+03
1.43
EPA-08
15
1.95E+06
1.61E+08
1.21
91.00
5.36E+03
1.70
EPA-09
15
2.10E+06
3.88E+08
0.54
64.79
3.93E+02
16.49
EPA-09
15
2.10E+06
3.88E+08
0.54
26.04
2.39E+03
1.09
EPA-10
15
3.90E+06
1.03E+09
0.38
108.00
1.52E+04
0.71
EPA-10
15
3.90E+06
1.03E+09
0.38
55.00
9.20E+03
0.60
EPA-13
15
1.73E+06
6.46E+08
0.27
13.90
2.19E+03
0.63
EPA-14
15
1.91E+06
2.10E+09
0.09
91.00
6.58E+03
1.38
EPA-14
15
1.91E+06
2.10E+09
0.09
56.00
1.97E+04
0.28
EPA-15
15
2.59E+06
3.61E+09
0.07
62.00
5.11E+04
0.12
EPA-15
15
2.59E+06
3.61E+09
0.07
45.00
1.74E+04
0.26
EPA-16
15
7.88E+05
1.31E+09
0.06
18.00
8.52E+03
0.21
EPA-16
15
7.88E+05
1.31E+09
0.06
21.00
4.91E+03
0.43
EPA-19
15
1.13E+06
8.32E+08
0.14
26.00
3.91E+03
0.66
EPA-19
15
1.13E+06
8.32E+08
0.14
28.00
2.42E+03
1.16
-------
Table 6. Viable Influenza Viral Particles Collected by NIOSH Samplers in a Calm-air Chamber
Experiment # Collect time Total aerosols PFU (in Viable viral Infectious conc. Normalized
(min) collected initial viral particles (PFU/L of air) infectious conc.
suspension)
collected (PFU)
(PFU/L of air)
collected
EPA-10
15
2.53E+09
3.90E+06
1.41E+03
26.90
14.59
EPA-10
15
2.53E+09
3.90E+06
1.48E+03
28.10
Average (SD)
15.23
14.91 (0.46)
EPA-05
30
4.07E+09
3.28E+06
1.60E+03
15.25
9.88
EPA-07
30
4.01E+09
1.61E+06
1.57E+03
14.96
20.05
EPA-08
30
4.85E+09
1.95E+06
1.64E+03
15.64
14.30
EPA-09
30
5.01E+09
2.10E+06
1.14E+03
10.87
8.93
EPA-09
30
5.01E+09
2.10E+06
8.75E+02
8.33
6.84
EPA-10
30
3.82E+09
3.90E+06
2.71E+03
25.83
15.01
EPA-10
30
3.82E+09
3.90E+06
3.15E+03
30.00
17.44
EPA-14
30
4.98E+09
1.91E+06
1.54E+03
14.64
13.29
EPA-14
30
4.98E+09
1.91E+06
2.06E+03
19.64
17.83
EPA-15
30
3.99E+09
2.59E+06
1.59E+03
15.12
12.68
EPA-15
30
3.99E+09
2.59E+06
1.59E+03
15.12
12.68
EPA-16
30
4.72E+09
7.88E+05
5.91E+02
5.63
13.10
EPA-16
30
4.72E+09
7.88E+05
5.17E+02
4.92
11.45
EPA-16
30
4.72E+09
7.88E+05
5.13E+02
4.88
11.36
EPA-16
30
4.72E+09
7.88E+05
6.80E+02
6.48
15.08
EPA-19
30
2.96E+09
1.13E+06
1.14E+03
10.83
Average (SD)
28.16
14.26 (5.01)
EPA-06
60
7.83E+09
4.80E+06
7.42E+03
35.35
17.38
EPA-08
60
8.96E+09
1.95E+06
2.60E+03
12.37
13.07
EPA-08
60
8.96E+09
1.95E+06
2.35E+03
11.19
11.83
EPA-08
60
8.96E+09
1.95E+06
2.24E+03
10.65
11.26
EPA-09
60
8.85E+09
2.10E+06
1.76E+03
8.37
8.32
EPA-09
60
8.85E+09
2.10E+06
1.63E+03
7.74
Average (SD)
7.69
11.59(3.51)
-------
Effect of Collection Medium on the Performance
of the NIOSH Sampler: The experimental results
showed that the NIOSH sampler is not as efficient at
maintaining viral viability as the SKC sampler. Since
virus is collected directly in liquid media with the SKC
sampler, we suspected that prolonged drying of the
collected virus, onto the walls of the collection tubes or
onto the backup filter with the NIOSH sampler, could
reduce viability. Therefore, the collection enviromnent
was modified as follows to address this issue:
Collection into supplemented HBSS: InEPA-04
which used the 1-jet collision nebulizer, one of the three
NIOSH samplers contained no collection media and
the other two had 1 or 5 ml of supplemented HBSS in
the Stage 1 tubes and 0.5 ml of supplemented HBSS
in the Stage 2 tubes. No plaques were observed in the
Stage 1 tubes of any of the three samplers (data not
shown), which suggests that the addition of 1 or 5 ml of
supplemented HBSS in the tubes did not improve the
collection efficiency presumably because the majority
of viral particles were deposited at the top of the
collection tubes and cannot reach the collection media
in the lower portion of the tubes. Therefore, in EPA-
19, we attempted to improve recovery and viability by
increasing the amount of supplemented HBSS to either
10 ml or 12 ml in Stage 1 tubes; 0.5 ml supplemented
HBSS was also added to the Stage 2 tubes. The results
(Figure 3) show that NIOSH samplers, with or without
supplemented HBSS, collected similar amounts of total
viral particles but viability (assessed using the VPA) was
reduced by an average of 54.4 % in tubes containing
supplemented HBSS.
ra
4-
o
_i
D
LI.
Q.
30.00 -
25.00 -
20.00 -
15.00
10.00
5.00 ¦
2.50E+04
I
¦
no medium Tl: 12 ml HBSS; Tl: 12 ml HBSS; Tl: 10 ml HBSS; Tl: 10 ml HBSS;
T2: 0.5 ml HBSS T2: 0.5 ml HBSS T2: 0.5 ml HBSS T2: 0.5 ml HBSS
o 2.00E+04
1.50E+04
Q- 1.00E+04
ro
5.00E+03
0.00E+00
no medium Tl: 12 ml HBSS; Tl: 12 ml HBSS; Tl: 10 ml HBSS; Tl: 10 ml HBSS;
T2: 0.5 ml HBSS T2: 0.5 ml HBSS T2: 0.5 ml HBSS T2: 0.5 ml HBSS
Figure 3. Effect of supplemented HBSS on the collection of viable and total viral particles.
14
-------
Collection into tubes coated with 2% agar in
supplemented HBSS: In EPA-07, the top portion of the
stage 1 tube was coated with 2% agar in supplemented
HBSS, either by aerosolization or manually. One of
the three NIOSH samplers had no coating on the stage
1 tube, one sampler's stage 1 tube was coated with
aerosolized agar, and another sampler's stage 1 tube
was coated manually with agar. Results demonstrated an
average decline in viability of 48% in the tubes coated
with agar (Figure 4).
25.00
(Q
M-
o
20.00
15.00
= 10.00
Q.
no coating T1 aerosolized agar T1 manually agar
coating
coating
2.50E+04
•5 2.00E+04
M-
o
v 1.50E+04
u
^ 1.00E+04
10
i-
'>
I 5.00E+03
0.00E+00
no coating T1 aerosolized agar T1 manually agar
coating coating
Figure 4. Effect of 2% agar coating on the collection of viable and total viral particles.
15
-------
Collection into tubes coated with 0.1% mucin:
Influenza strains bind sialyloligosaccharides in mucin on
tracheal epithelium (11). In EPA-15, the top portion of
the stage 1 tube was coated manually with a 0.1% mucin
solution. The stage 1 tubes in two NIOSH samplers had
no coating, and two NIOSH samplers contained coated
stage 1 tubes. The results (Figure 5) showed that the
NIOSH samplers with and without coating collected
essentially the same amount of viable and total viral
particles (average 13.22 versus 12.68 PFU/L of air in the
coated and non-coated tubes, respectively; average 1.51
X 104versus 1.46 X 104 total viral particles/L of air in
the coated and non-coated tubes, respectively).
15.00 -|
14.00 -
™ 13.00 -
o
2 12.00 -
11.00 -
10.00
no coating
no coating T1 coated with T1 coated with
0.1% mucin
solution
0.1% mucin
solution
re
o
1.80E+04 -i
1.70E+04 -
1.60E+04 -
ju 1.50E+04 -
u
1.40E+04 H
re
a.
"!5
"!5
4-*
o
1.30E+04 -
1.20E+04 -
1.10E+04 -
1.00E+04
no coating no coating T1 coated with T1 coated with
0.1% mucin 0.1% mucin
Figure 5. Effect of 0.1% mucin coating on the collection of viable and total viral particles.
16
-------
Development of ail Alternative Viability Assay: To
increase sensitivity and improve detection of viable
virus, a viral replication assay (VRA) based on a
coupled TCID50/qPCR assay was developed. In EPA-
17 and EPA-IS, MDCK cells were infected with the
viral samples collected by the NIOSH samplers and the
SKC samplers and after 24 h of incubation, the cells
were lysed and MDCK cell infection was detected by
qPCR analysis. The results from EPA-17 (data not
shown) demonstrate high viable viral loads in samples
collected from both the NIOSH samplers (total mean
2.1 X 10s virus/L of air) and SKC samplers (total mean
6.4 X 10s virus/L of air). This result supports the above
data obtained by the VPA and the concept that viable
influenza A can be carried in aerosolized particles.
Coating of electret filters to the Stage 1 and Stage
2 tubes-Since virus is deposited at the tops of the
collection tubes, it was possible that the amount of
supplemented HBSS added to the collection tubes
was still not enough to reach the point of deposition.
However, airflow of the sampler would be severely
compromised with a further increase in supplemented
HBSS to the tubes. As an alternative, in EPA-IX,
electret filters (12) wetted with supplemented HBSS
were inserted inside the top portion of the stage 1 and
2 collection tubes in order to collect virus directly into
the liquid environment of the filters. The results (Figure
6) showed that the number of virus collected from the
sampler containing the electret filters was about the same
as that collected in the absence of the filters (6.3 X 105
versus 8.0 X 105, respectively) but the distribution of
virus was significantly affected. The number of virus
collected in stage 1 (>4 |im particles) and stage 2(1-4
|im particles) increased 25% and 64%, respectively,
while the amount of virus on the filter (<1 |im particles)
decreased 73%. The VRA results, however, revealed a
further 67% decrease in viral viability in the presence of
the electret filters. The reduced viability of the collected
virus may be the result of drying of the electret filters
during the sample collection. A method to keep the filters
wetted during sampling is under consideration.
-------
viral replication assay
4.00E+10
2.00E+10
0.00E+00
pCR for total viral particles
6.00E+05
4.00E+05
2.00E+05
¦ no electret filter
0.00E+00
viral replication assay
total # M copies
T1 plus electret filter
1.81E+09
T2 plus electret filter
5.20E+09
IF
9.46E+08
| T1 no electret filter
1.87E+09
| T2 no electret filter
1.24E+10
53F
2.83E+10
qPCR for total viral particles
total # M copies
| T1 plus electret filter
8.77E+04
T2 plus electret filter
4.10E+05
IF
1.31E+05
["esiftfts
| T1 no electret filter
7.01E+04
T2 no electret filter
2.50E+05
53F
4.78E+05
r7.5S5il
Figure 6. Effect of electret filter coating on the collection of viable and total viral particles.
18
-------
Effect of storage time on the samples collected
by NIOSH samplers and SKC samplers: In field
studies, airborne influenza viral particles collected
by samplers are generally stored at 4°C for up to 24 h
before processing. To investigate whether influenza
viral samples remain viable when stored at 4°C prior
to processing, we conducted EPA-14 in which four
NIOSH samplers and two SKC samplers were used to
collect the viral particles. After collection, the samples
in two NIOSH samplers were immediately extracted and
assayed. The samples in the other two NIOSH samplers
were immediately resuspended in supplemented HBSS
and then stored at 4°C for 24 h before processing. For
the SKC samples, half of the extracted sample from each
sampler was processed immediately and the other half
was stored at 4°C for 24 h before processing. The results
(Figure 7) showed that 24 h storage at 4°C does not
significantly affect viral viability.
D
Ll_
Q.
120.00 -|
100.00 -
80.00 -
60.00 -
40.00 -
20.00 -
0.00 -
¦
NIOSH (analyzed NIOSH sampler SKC (analyzed SKC (+4C O/N
imme.) (+4C O/N storage) imme.) storage)
3.50E+04 -|
± 3.00E+04 -
is
° 2.50E+04 -
V)
-5 2.00E+04 -
t
Q. 1.50E+04 -
(O
2 1.00E+04
B 5.00E+03 -
0.00E+00 -
_JJ
NIOSH (analyzed NIOSH sampler SKC (analyzed SKC(+4CO/N
imme.) (+4C O/N storage) imme.) storage)
Figure 7. Effect of storage time on the samples collected by NIOSH and SKC samplers
19
-------
Distribution of collected airborne influenza virus in
different sampling stages: The particle sizes and con-
centrations of aerosols loaded in the calm-air chamber
were monitored by an aerodynamic particle sizer (APS)
for each experiment. A typical particle size distribution
of aerosols (mass based) loaded in the calm-air chamber
is shown in Figure 8. The mass median aerodynamic
diameter was 1.0 |im. Our previous studies (3) showed
that when chamber air loaded with viral-laden aerosols
is drawn into an inlet at 3.5 L/min, particles with diam-
eter larger than 4 |im are collected in the Stage 1 tube
of the NIOSH sampler, 1-4 |im particles are collected
in the Stage 2 tube, and particles with diameter smaller
than 1 |im are collected on a backup filter. The average
distribution of collected viable and total viral particles in
the collection tubes and backup filter from all calm air
chamber experiments is shown (Table 7). Eighty-six per-
cent of viable particles (mean of the samples collected at
three collection times) were contained in aerosols with
diameters < 4 |im. When calm air experimental results
are averaged, the distribution of the viable viral particles
in the two stages and on the backup filter was similar re-
gardless of collection times though the distribution of the
collected total viral particles appeared to vary with collec-
tion times. When compared to the 15 min collection time,
the fraction of the collected total viral particles after a 60
min collection decreased from 21% to 3% in the Stage 1
tubes and increased from 37% to 63% on the backup filter
(Table 7).
120 -|
100 -
80 -
60 -
40 -
20 -
0
0 5 10 15 20
Aerodynamic Diameter (um)
25
VP
ON
£
o
'+¦»
u
50.00
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
Mass Median Aerodynamic
Diameter: 1.0 um
1 I I I I I I I I I I I I I I I I I I
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
i
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
V
o
t-H
(N
ro
LO
to
r<
00
cri
i
i
i
i
i
i
i
i
T—1
i
Figure 8. Atypical size distribution of aerosols in the calm-air chamber.
20
-------
However, examination of individual experiments
emphasizes the variability observed among experiments.
This can be seen, for example, by comparing EPA-08
and EPA-09 where collection occurred for 30 and 60
mill in each experiment, with EPA-10 where collection
occurred for 15 and 30 mill (Table 8). Within the same
experiment, the samplers with different collection times
showed less variability when comparing the distribution
of viable with total viral particles, indicating that the
NIOSH samplers performed consistently over time
within a given experiment. In contrast, in EPA-10 a
higher fraction of the total viral particles was found in
the Stage 1 tubes than that found in EPA-08 and EPA-
09. This variation may be the result from using different
concentrations of the initial viral stocks for generation
of the airborne influenza viral particles. EPA-10 had
6.4 times more viral particles in the nebulizer solution
than EPA-08, which may have led to a concentration-
dependent distribution of viral particles.
Table 7. Distribution of Collected Viable and Total Airborne inftuen/.a Virus in the Stage 1, Stage 2 and Backup
Filter of the NIOSH Samplers (Average of all the NIOSH Samplers U sed in the Project)"
Collection
Distribution of collected viable influenza virus
Distribution of collected total viral particles
time (min)
T1
T2
F
T1
T2
F
15
19(1)%
55(2)%
26(1)%
21(3)%
42(2)%
37(1)%
30
12(5)%
43(11)%
45(12)%
11(7)%
31(12)%
58(13)%
60
11(6)%
48(9)%
41(11)%
3(2)%
34(5)%
63(7)%
" The number in parentheses is the standard deviation.
Table 8. Distribution of Collected Viable and Total Airborne Influcn/.a Virus in the Stage 1, Stage 2 and Backup
Filter of the NIOSH Samplers (The Individual Experiments for the Effect of Collection Times)
Experiment #
Sampler stages
30 min
60 min
Viable
T otal
Viable
Total
EPA-08
T1
7.91%
4.18%
13.60%
3.3%
T2
57.08%
32.13%
52.11%
36.24%
F
35.01%
63.70%
34.29%
60.46%
EPA-09
T1
11.42%
0.33%
9.29%
0.81%
T2
35.72%
20.63%
39.84%
28.61%
F
52.86%
79.03%
50.87%
70.58%
Experiment #
Sampler stages
15 min
30 min
Viable
Total
Viable
T otal
EPA-10
T1
18.60%
21.34%
22.11%
25.12%
T2
55.01%
41.82%
51.19%
39.83%
F
26.39%
36.84%
26.70%
35.05%
21
-------
22
-------
4.0
Discussion
Concerns regarding the vulnerability of large populations
to potentially deadly pathogens such as viruses, and
the emergence of pandemic strains of influenza A, have
prompted a number of studies into the mechanisms of
their transmission (13-20). Of the various modes of
transmission possible, aerosol transmission poses a
profound threat since it has the greatest potential for
widespread dissemination. Acrosoli/ed virus in particles
less than 4 juii in diameter can be easily inhaled into the
alveolar region of the lungs and can also remain airborne
for an extended period of time (4). Recent studies
revealed that 42-53% of influenza virus in healthcare
facilities was associated with airborne particles less than
4 jun in si/c. but whether the viability of the virus was
not determined (2,21). There arc a number of aerosol
samplers in use. yet these vary greatly in their efficiency
of collection and in their ability to maintain the viability
of collected viruses. In addition, the determination of
the magnitude of a potential epidemic would require a
determination of the presence of infectious acrosoli/ed
virus. The presence of infectious acrosoli/ed virus, in
turn, would requires si/e-fractionation. However, none
of the samplers can size-fractionate particles. Therefore,
the technical limitations of the samplers interfere with
the ability to determine the magnitude of a potential
epidemic.
In a recent study (1), the efficiencies of four commercial
air samplers were compared. In tliat study, the SKC
sampler was superior to 37 mm cassette samplers
containing cither a Teflon filter (74% recovery) or a
gelatin filter (63% recovery), and the CCI sampler that
contains a polyurcthane foam filter (32% recovery). The
NIOSH sampler's efficiency at collecting acrosoli/ed
particles for 30 mill from a calm-air chamber is
essentially the same as that from the SKC sampler
that collects particles directly into a liquid media
(1.2 X 104 TVP/L of air versus 1.3 X 104 TVP/L of
air, respectively). Further, the efficiency is relatively
constant over the collection times of 15, 30, and 60 mill.
In the initial assessment of the NIOSH sampler, the
efficiency of extracting virus that had been spiked
directly into the Stage 1 collection tube and backup filter
was addressed. On average, 11% of the virus could be
recovered from the filter and 15.8% could be recovered
from the Stage 1 tube. The viability of virus extracted
from the filter and the Stage 1 tube was 56.7% and
58%, respectively. These results arc in stark contrast to
the >95% recovery of virus that was first acrosoli/ed
and collected by the NIOSH sampler. Fabian ct al. (1)
reported a similar finding in that particle extraction
efficiency of spiked or acrosoli/ed virus from a
particular sampler was not consistent. They emphasized
the importance of conducting sampler experiments using
test aerosols rather than spiking experiments.
The SKC sampler is reported to be the most efficient
aerosol sampler at maintaining essentially 100% viability
of collected virus (1). In contrast, recoveries of viable
virus from samplers with a gelatin filter. Teflon® filter,
or polyurcthane filter, were only 10%, 7%, and 22%,
respectively (1). The NIOSH sampler maintains viable
recovery at an average 26% and. thus, surpasses all but
the SKC sampler. In this study, the viability of virus in
the initial suspension used to generate the acrosoli/ed
particles was only 0.06-1.43% (average 0.53%) of the
total viral particles. Viral stocks with similarly low (0.3-
0.5%) viability were used in the study by Fabian ct al.
(1). Conceivably the viability is actually much higher a.s
viability assessed by the chicken embryo infectious dose
endpoint 50% (CEID50) from the manufacturer (ATCC)
is 10-50X higher. This discrepancy may be due to a
lack of sensitivity of the VPA for determining viability
or due to the decline in stock viability with storage
time. Whatever the cause, there is a need to increase
the sensitivity of the viability assessments for future
studies. To address this issue, a VRA based on a coupled
TCID50/qPCR assay to amplify viral particle number and
increase sensitivity was developed in the later stage of
this project. The VRA was used to analyze the initial
viral solutions and the aerosol samples in ERA. 17 and
EPA 18. The results showed highly viable viral loads
in the viral suspensions and the samples, yet how the
viable viral loads detected by the VRA relate to the
actual number of viable virus prior to viral replication is
unknown. This is because the VRA detects virus tliat has
infected and replicated from an unknown copy number
within MDCK cells during the 24 h incubation period.
The amount of virus per cell is expected to exponentially
increase and then plateau at some point during the 24 h
incubation. Tliercforc. the number of viral replications/
cell is not easily determined. Additionally, virus
secreted from an infected MDCK cell can subsequently
infect MDCK cells that were not initially infected,
further complicating the quantification of starting virus
in each collection sample. Further studies arc needed to
correlate the number of total viral particles collected and
detected by the qPCR with the viability results of the
VRA. In the future, an assay with sufficient sensitivity,
and a viral suspension with a large percentage of viable
23
-------
viral particles for ncbuli/ation. will certainly reduce the
experimental uncertainties and improve viability studies
on airborne vims.
A previous study on collection of viable airborne vims
(1) has suggested that samplers containing collection
medium can preserve the viability of collected vims
better than those without collection medium. The
NIOSH sampler retains viability at 26% of the total
viral population; however, we attempted to improve this
by collecting viral particles into a moist environment.
To this end. we added supplemented HBSS media
to the collection tubes. We found that the addition
of supplemented HBSS did not improve viability,
possibly because the vims is deposited at the top of the
tube and above the media rather than into the media.
Adding more media, however, would negatively alter
the aerodynamics of collection. Alternatively, the
collected viral particles may have become too dilute
and the sensitivity of the plaque assay may have been
compromised. Diverting the deposition of viral particles
to a lower part of the collection tube would reduce the
amount of supplemented HBSS needed for collection,
without adversely altering the aerodynamics of the
sampler. To circumvent the above problems, the top
portion of the Stage 1 collection tubes were coated
with 2% agar or 0.1% mucin, or supplemented HBSS-
soaked electrets filters were inserted near the top of the
tubes. However, neither viability nor the total number of
recovered viral particles increased over the tubes lacking
these materials. It is possible that the extraction of vims
from these coatings was insufficient and. as a result,
the coatings dried-out during the collection period.
Alternatively, the extraction of vims could have been too
harsh and. as a result, viability was negatively affected.
The results from these studies support the use of the
NIOSH sampler for analysis of air quality in an outside
environment or within occupational environments such
as hospital emergency rooms. Analyses of outside
air for aerosolized vims may be complicated by the
presence of other microorganisms, dust, or pollen that
may alter the aerodynamics of fractionation or provide
a hitch-hiking mode of transport for the vims. We
have investigated whether the NIOSH sampler can
fractionate co-acrosoli/ed particles. We showed that,
while efficient separation of influenza A and Aspergillus
versicolor fungal spores was possible, there was a
shift in the overall deposition of vims to the Stage 1
collection tube, and fewer viral particles were found
on the backup filter (3). Further, other environmental
parameters such as humidity likely influence whether
fractionation is shifted or whether viable vims is
recovered. An early study showed that the stability of
influenza A is minimal at 50% relative humidity (RH),
high at 60-80% RH. and maximal at 20-40% RH (22).
A more recent study essentially confirmed those results
and showed tliat transmission of influenza docs not
occur at 80% RH. is low at 50% RH, high at 65% RH.
and maximal at 20% and 35% RH (23). The stability of
other viruses including Seniliki forest vims (24), HIV
(25), and respiratory syncytial vims (26) were shown
to be affected by the RH. For this study, we maintained
the RH at 20% for all experiments; future work would
include an investigation into the role of humidity on
viability.
An important distinction between the NIOSH sampler
and the SKC sampler is the ability to fractionate
aerosols and identify which fractions contain viable
vims. Coughing, sneezing, and talking generate airborne
particles ranging in si/c from a few millimeters to less
than one micrometer. Particles less than 10 jini arc,
arguably, the most problematic as they can remain
airborne for hours and arc readily inhaled deeply into
the respiratory tract. Know ing whether viable influenza
A is present on tliese small particles provides better
assessments for risk of infection and precautionary
guidelines for prevention. Such information would
dictate the type of particle mask or respirators to use.
enable appropriate adjustments to air handling systems,
and determine which aerosol-generating medical
procedures to avoid during influenza outbreaks. Unlike
the SKC BioSanipler, the NIOSH sampler is easier for
people to wear as a personal sampler because it docs not
require aqueous buffers for capturing airborne vims and.
therefore, potential spillage onto clothing is eliminated.
Additionally, field use is simplified as the collection
tubes of the NIOSH sampler can be capped and stored
after air sampling, and vims deposited on the dry walls
of the tubes can be later recovered into liquid media.
In contrast, vims collected directly into liquid media is
potentially more labile and the samples would need to be
maintained at cooler temperatures. Lastly, air sampling
with the NIOSH sampler occurs at 3.5 L/iiiin versus 12.5
L/iiiin with the SKC sampler, and, thus, a smaller and
lighter sampling pump could be worn.
-------
5.0
Conclusions
The use of the NIOSH aerosol sampler for investigation
of real-world environmental samples holds much
promise. The sampler is a lightweight device that
could be used either as an area sampler (i.e. hospital
room), or as a personal breathing zone air-sampler that
could be worn on the clothing of healthcare workers or
others. The NIOSH sampler eliminates sample loss, and
minimizes the sample contamination and degradation
that is associated with most other aerosol samplers. The
demonstrated ability of the sampler to collect a viable
surrogate virus, influenza A, leads us to be cautiously
optimistic about its potential use in the detection of other
viruses and microorganisms. Increased acceptance of
the NIOSH sampler as a better alternative to other air
samplers requires improvements in viability retention,
particularly from the backup filter that collects the
smallest particles, i.e., those most likely suited for long-
range dissemination and deep lung inhalation.
25
-------
26
-------
6.0
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