methods for sampling of airborne virusesmethods for sampling of airborne viruses daniel verreault,1...
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MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2008, p. 413–444 Vol. 72, No. 31092-2172/08/$08.00�0 doi:10.1128/MMBR.00002-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Methods for Sampling of Airborne VirusesDaniel Verreault,1 Sylvain Moineau,2,3 and Caroline Duchaine1,2*
Centre de Recherche, Hopital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l’Universite Laval,2725 Chemin Ste.-Foy, Quebec City, Quebec, Canada G1V 4G51; Departement de Biochimie et de Microbiologie,
Faculte des Sciences et de Genie, Universite Laval, Pavillon Alexandre-Vachon, 1045 Avenue de la Medecine,Quebec City, Quebec, Canada G1V 0A62; and Groupe de Recherche en Ecologie Buccale (GREB)
and Felix d’Herelle Reference Center for Bacterial Viruses, Faculte de Medecine Dentaire,Universite Laval, Pavillon de Medecine Dentaire, 2420 rue de la Terrasse,
Quebec City, Quebec, Canada G1V 0A63
INTRODUCTION .......................................................................................................................................................413CONTACT VERSUS AEROSOLS............................................................................................................................413EPIDEMIOLOGICAL EVIDENCE OF AIRBORNE SPREAD OF VIRUSES ..................................................413COMMON SOURCES OF AIRBORNE VIRUSES ...............................................................................................414SIZE DISTRIBUTION OF VIRUS-LADEN PARTICLES ....................................................................................415FACTORS AFFECTING THE RECOVERY OF AIRBORNE VIRUSES BY SAMPLING ..............................416AIR SAMPLING METHODOLOGIES....................................................................................................................417AIR SAMPLING FOR VIRUS RECOVERY...........................................................................................................417
Solid Impactors .......................................................................................................................................................417Liquid Impactors ....................................................................................................................................................418Filters .......................................................................................................................................................................419Electrostatic Precipitators .....................................................................................................................................439Other Sampling and Detection Methods.............................................................................................................440
ASSESSING THE EFFICIENCY OF AIRBORNE VIRUS SAMPLERS BY USE OF TRACERS..................440LABORATORY STUDIES OF AIRBORNE VIRUSES .........................................................................................440SURROGATE VIRUSES............................................................................................................................................440CONCLUSIONS .........................................................................................................................................................441ACKNOWLEDGMENTS ...........................................................................................................................................441REFERENCES ............................................................................................................................................................441
INTRODUCTION
Any microorganism, including viruses, can become airborne.Contaminated material can be aerosolized in many differentways, ranging from wind to human and animal activities such assneezing, mechanical processes, etc. If the aerodynamic size ofan infectious particle is appropriate, it can remain airborne,come into contact with humans or animals, and potentiallycause an infection. The probability of an airborne microorgan-ism-laden particle causing an infection depends on its infec-tious potential and its ability to resist the stress of aerosoliza-tion.
Airborne microorganisms can represent major health andeconomic risks to human and animal populations. Appropriatepreventive actions can be taken if the threat posed by suchmicroorganisms is better understood. Authorities need to beaware of the nature, concentration, and pathogenicity of air-borne microorganisms to better control them. This informa-tion can be obtained by using various air sampling methods,each of which has its particular advantages and disadvantages.Many types of samplers and analytical methods have been usedover the years (Fig. 1). The purpose of this review is to present
the principles underlying viral aerosol sampling methods, withtheir advantages and pitfalls.
CONTACT VERSUS AEROSOLS
The route of transmission of infections is not always easilydetermined in an environment with undefined parameters. In-fection by direct contact can occur when infected hosts are inclose proximity with a susceptible population. On the otherhand, infected hosts can transmit the disease without directcontact. Moreover, many microorganisms, including viruses(110), can remain infectious outside their hosts for pro-longed periods of time, and this can lead to infections byindirect contact. For example, a surface can become con-taminated by deposited infectious droplets and eventuallycause the infection of susceptible hosts coming into contactwith it. The probability of airborne transmission of an in-fectious disease can be determined by conducting epidemi-ological studies (145) and/or by analyzing the microbiolog-ical content of air samples.
EPIDEMIOLOGICAL EVIDENCE OF AIRBORNESPREAD OF VIRUSES
Studies on the aerobiology of infectious diseases, includingviral diseases, have been rather limited (115). This is duemainly to the difficulty in collecting and analyzing airbornebiological contaminants, which is an even greater problem forviruses. This technical challenge has made epidemiological
* Corresponding author. Mailing address: Centre de Recherche,Hopital Laval, Institut Universitaire de Cardiologie et de Pneumolo-gie, 2725 Chemin Ste.-Foy, Quebec City, Quebec, Canada G1V 4G5.Phone: (418) 656-8711, ext. 5837. Fax: (418) 656-4509. E-mail: [email protected].
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studies particularly useful. While data inferred from epidemi-ological studies using computer-based analytical methods aremore equivocal than those from air sampling coupled withmicrobial analyses, epidemiological studies can provide veryvaluable information.
Many epidemiological studies have proposed that virusescan spread from one host to another by using air for transport.The capacity of the foot-and-mouth disease (FMD) virus tospread by air has been studied and reviewed (36) over the yearsand is now being investigated using computer models. One ofthese models predicted that in a “worst-case scenario” of anFMD outbreak, cattle could be infected as far as 20 to 300kilometers away from an infectious source (37). Dispersionmodels based on meteorological data and information on thespread of FMD at the beginning of the 1967–1968 epidemic inthe United Kingdom strongly suggested that the infection mayhave spread by the airborne route over a distance of 60 km(59). Airborne transmission of FMD was also reported to haveoccurred during the 1982–1983 epidemic in Denmark. In thelatter case, an analysis of epidemiological dynamics using mo-lecular methods coupled with meteorological data concludedthat the infection had spread by air over a distance of 70 km(27). Similarly, the results of a Canadian study on an FMDepidemic reported that airborne viruses may have traveled 20km downwind from the contaminated source (29). Neverthe-less, a recent study on the O/UKG/2001 strain of FMD virusindicated that it does not spread efficiently between sheep bythe airborne route. However, other strains may behave differ-ently (134).
In 2001, a Norwalk-like virus outbreak in a school in theUnited Kingdom was believed to have been caused by airbornetransmission (89). A similar occurrence has also been reported
for a hotel restaurant (88). A retrospective cohort study con-ducted after a severe acute respiratory syndrome (SARS) ep-idemic in Hong Kong in 2003 suggested that airborne spreadmay have played an important role in the transmission of thedisease (146). The same mode of transmission was also hypoth-esized in other studies of SARS (87, 104, 145). Aerosols mayalso be responsible for the transmission of other viral diseases(63, 83, 113).
COMMON SOURCES OF AIRBORNE VIRUSES
A virus can multiply only within a host cell. Infected cells canspread viruses directly into the surrounding air (primary aero-solization) or to fluids and surfaces, which can become sourcesfor airborne transmission (secondary aerosolization). Second-ary aerosolization can occur for any virus, predominantly whenair displacements or movements around contaminated surfacesor fluids disperse the viruses into the air. It can also occur byliquid splashes, which can aerosolize viruses in liquids or onsurfaces. In fact, almost any kind of disturbance of infectedorganisms or materials, even the bursting of bubbles in seawa-ter (9), can produce airborne, virus-laden particles.
The most important aerosol source representing a risk forhuman health is humans themselves. Since the interspeciesbarrier is not a factor in the transmission of infections fromhuman to human, aerosol-mediated infections from humansources can occur in everyday situations. Human infectionsthrough viral aerosol sources have been studied in variousenvironments, including office buildings (102), hospitals (3, 10,11, 13, 19, 41, 92, 95, 117, 126), restaurants (88), and schools(89). The mechanisms of dispersion of infectious aerosols orig-inating from humans are described in detail in a recent review
FIG. 1. Airborne virus sampling studies, according to date and analysis method.
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(98). It is important to recognize that viruses can be spread byairborne particles released by humans but also by other means.Simply flushing a toilet containing infectious particles canaerosolize significant concentrations of airborne viruses (14,136). Wastewater treatment plants (24, 51) and sewage sprin-klers (97, 125) can also produce viral aerosols.
Farm animals have also been studied for their emission ofairborne viruses. The FMD virus, which is one of the mostwidely studied airborne animal viruses, has been detected in aircontaminated by infected pigs and ruminants (7, 8, 38, 39, 56,60, 121) in both laboratory settings and farm environments.This single-stranded RNA (ssRNA) virus of the Picornaviridaefamily is excreted in all body fluids of infected animals (100)and can become airborne directly from the animals or from thesecondary aerosolization of deposited viruses or virus-ladenparticles. Other suspected sources of airborne viruses, such asburning carcasses of infected animals (26), have not yet beenidentified formally as true sources because additional investi-gations are needed.
Poultry farms are also potential producers of virus-ladenairborne particles. The exotic Newcastle disease virus(Paramyxoviridae family) was probably the first virus isolatedfrom a naturally contaminated environment (35) of poultryhouses sheltering infected birds. This 150-nm-diameter ssRNAvirus was detected in air samples from two farms during anoutbreak in Southern California in 2002–2003 (74). Air sam-ples in and around broiler poultry houses have also been stud-ied for the presence of viruses such as Escherichia coli bacte-riophages, which are a fecal contamination tracer (50). Otheranimals, such as bats (rabies virus) (144), rabbits (rabbit pox-virus) (128, 141), and mice (polyomavirus) (94), have beenstudied as sources of bioaerosols. These viruses can be releasedinto the air directly from animals by their breathing, coughing,and sneezing or by secondary aerosolization. It should benoted that the means of aerosolization has a critical impact onthe aerodynamic size and, thus, on the behavior of the airborneparticles.
SIZE DISTRIBUTION OF VIRUS-LADEN PARTICLES
For humans, most particles larger than 10 �m will not passthe upper airways; while smaller particles will travel moreeasily toward the lungs, the particles will be trapped at differentproportions in the head airways and the tracheobronchial andalveolar regions (75). The particle size determines whether ornot it can be inhaled and retained in the respiratory tract.
Given that virus-laden particles are a complex mixture ofvarious components (salts, proteins, and other organic andinorganic matter, including virus particles), it is essential torealize that the size of the viral particle itself does not rule theairborne particle size. The influence of viruses alone on thegranulometric distribution of aerosols is likely negligible com-pared to that of the remainder of the aerosol. To support this,it was demonstrated that the particle size distribution of arti-ficially produced submicrometer and ultrafine aerosols of cul-ture media is not affected by the presence of bacteriophages(76).
Infectious bioaerosols spontaneously released by sick ani-mals are composed of variously sized particles. The smallersize limit of a viral aerosol is limited to the virus diameter itself,
which can be as small as 20 to 30 nm, while the larger limitdepends on the size of the particle with which it is associated.Size also dictates the capacity of a particle to remain airborne.A study investigating the natural excretion of the FMD virus(25 to 30 nm in diameter) into the air by infected pigs, using amultistage liquid impinger sampler, showed that 65% to 71%of the virus-laden particles were over 6 �m in diameter, 19% to24% ranged from 3 to 6 �m in diameter, and 10% to 11% wereunder 3 �m in diameter (121). Similar results were also ob-tained with infected sheep (56). The same type of bioaerosolsampler was also used to establish a link between the concen-tration and the size of infectious particles, using artificially andnaturally produced aerosols of FMD virus. This study reportedthat over 85% of the particles in the artificially produced aero-sols were less than 3 �m in diameter, whereas the size distri-butions of the natural aerosols were similar in all three stagesof the sampler (39). Another study investigating pigs infectedwith Aujeszky’s disease virus (Herpesviridae family; approxi-mately 150 nm in diameter; double-stranded DNA [dsDNA]virus) found that the infectivity of the aerosols collected ineach stage of the three-stage impinger varied over time. Theinvestigators reported that the size distributions of the aerosolsin the three stages were comparable on day 2 of the infectionbut that there was an increase in infectivity associated withlarger particles on days 3 and 4 (40). Nevertheless, no clearassociation has been made between aerosol infectivity and aparticular size range (60).
While single virus particles exist in the air (76), they tend toaggregate rapidly. Aggregation speed depends on the size dis-tribution of the airborne particles, the concentration of theaerosol, and the thermodynamic conditions (142). Many fac-tors influence the size distribution of both naturally and arti-ficially produced viral aerosols. Artificially produced aerosolsare normally used in controlled environments where there areno other aerosols to which the nebulized particles can bind.They are thus influenced only by the size of the original dropletcreated by the nebulizer and by the solute concentration inthe droplet. When a droplet evaporates (Fig. 2), its final size (thedroplet nucleus) depends on the relative humidity (RH) in thechamber. To some extent, this phenomenon can also be ob-served with natural aerosols. For example, infectious dropletsexhaled by animals shrink rapidly with the lower humidityoutside the respiratory airway, creating smaller aerosols. How-ever, the size distribution of such naturally generated bioaero-sols depends on the sizes of the particles to which the micro-organisms bind. This binding may occur by diffusion,impaction, interception, or electrostatic attraction (98). Ifmostly large particles are encountered in the air of a givenenvironment, then the particles making up the infectious aero-sol will also tend to be large. Interestingly, larger particles maybe relatively less hazardous than smaller ones. It has beenshown on pig farms that a visually clean environment may bemore contaminated by bioaerosols than a visually dirty one(43). This may be due to the fact that larger particles tend tosettle faster than smaller particles do; the settling velocity of0.001-�m particles is 6.75E�09 m/s, while 10-�m particlessettle at 3.06E�03 m/s and 100-�m particles settle at2.49E�01 m/s (75). Airborne particles in a “clean” environ-ment are more likely to remain small and inhalable by animals
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and humans than are particles in a dirty environment, whichtend to grow larger by sticking to other airborne particles.
FACTORS AFFECTING THE RECOVERY OF AIRBORNEVIRUSES BY SAMPLING
Organic and inorganic materials in viral aerosols can affectthe size of the aerosolized particles and their infectious poten-tial. Many factors, such as RH, temperature, radiation, aero-solization medium, exposure period, chemical composition ofthe air, and sampling methods, can affect the infectivity ofairborne viruses. Each virus reacts in its own particular way toeach factor or combination of factors, depending on the struc-tural composition of the virus and its interactions with otheraerosol components. However, the structural composition ofairborne viruses alone cannot be used to predict survival underdifferent environmental conditions (78).
RH is the most widely studied of the factors that affectairborne virus infectivity (Table 1). Depending on the virus,optimal preservation of infectivity may require a low RH (un-der 30%), an intermediate RH (30% to 70%), or a high RH(over 70%). Influenza virus (65, 118), Semliki Forest virus (17),Japanese B encephalitis virus (86), porcine reproductive andrespiratory syndrome virus (72), Newcastle disease virus, and
vesicular stomatitis virus (122), all of which are enveloped, aremost stable at low RH, while rhinovirus (79, 84), poliovirus (65,79, 81), T3 coliphage (45, 122), rhinotracheitis virus (122),picornavirus (5), and viruses of the Columbia SK group (4),which are nonenveloped (with the exception of the rhinotra-cheitis virus), are most stable at high RH. Human coronavirus229E (79), pseudorabies virus (119), and rotavirus (81, 82, 116)are most stable at intermediate RH. The first two are envel-oped, while mature rotaviruses are usually nonenveloped. RHhas no effect on the stability of airborne St. Louis encephalitisvirus under the conditions tested (112).
Seasonal variations in indoor RH have also been correlatedwith fluctuations in the morbidity of influenza (low RH) andpoliomyelitis (high RH) viruses, with the highest morbidityoccurring at the optimal RH for each virus (68, 69). Seasonalvariations have also been observed with measles virus (34) andrespiratory syncytial virus (147). An intriguing study comparingthe effect of RH on the stability of an airborne picornavirus tothat on its genomic RNA (5) indicated that the inactivation ofairborne picornaviruses by low RH levels is not due to theinstability of the RNA but, rather, to structural damage to thevirion (5). The findings of these studies indicate that there is noabsolute correlation between RH and the preservation of viral
FIG. 2. Evaporation of a liquid droplet (left) to a droplet nucleus (right). As the liquid evaporates, the nonevaporative content concentratesuntil a droplet nucleus is obtained.
TABLE 1. Effects of RH on infectivity of a selection of airborne viruses
Virus Optimal RH formaximum infectivity Family Genetic material Size (nm) Envelope
Influenza virus Low Orthomyxoviridae ssRNA (�) 80–120 YesNewcastle disease virus Low Paramyxoviridae ssRNA (�) 150 YesVesicular stomatitis virus Low Rhabdoviridae ssRNA (�) 60 � 200 YesJapanese encephalitis virus Low Flaviviridae ssRNA (�) 40–60 YesPorcine reproductive and respiratory
syndrome virusLow Arteriviridae ssRNA (�) 45–60 Yes
Semliki Forest virus Low Togaviridae ssRNA (�) 70 YesHuman coronavirus 229E Mid-range Coronaviridae ssRNA (�) 120–160 YesRotavirus Mid-range Reoviridae dsRNA 100 NoPseudorabies virus Mid-range Herpesviridae dsDNA 200 YesRhinovirus High Picornaviridae ssRNA (�) 25–30 NoPoliovirus High Picornaviridae ssRNA (�) 25–30 NoPicornavirus High Picornaviridae ssRNA (�) 25–30 NoColumbia SK group High Picornaviridae ssRNA (�) 25–30 NoT3 coliphage High Podoviridae dsDNA 60 (capsid) NoRhinotracheitis virus High Herpesviridae dsDNA 200 YesSt. Louis encephalitis virus All Flaviviridae ssRNA (�) 40–60 Yes
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infectivity in aerosols and that the impact of RH should bedetermined for each virus. However, it appears that low RHtends to preserve the infectivity of enveloped viruses, while thestability of nonenveloped viruses is best preserved at high RH.
Temperature can also have an impact on the infectivity ofairborne viruses. For example, the stability of certain infectiousairborne viruses (47, 65, 72, 79, 119, 122) is improved at lowtemperatures but does not depend on RH. UV radiation isanother factor that influences survivability. UV germicidallamps, for instance, can be used to inactivate airborne micro-organisms, including viruses, in indoor settings (53). However,in certain cases, RH must be taken into consideration. Forexample, vaccinia virus is more susceptible to UV radiation atlow RH than at high RH (93).
Interestingly, aerosolization can inactivate some viruses to acertain extent, depending upon the nature of the spray fluid,the temperature, and the RH (80). This was reported for therecovery of bovine parainfluenza virus (47) and infectious bo-vine rhinotracheitis virus, where various combinations of thesefactors generated different results (48). Certain chemicals alsohave diverse effects on the stability of airborne viruses. Forexample, adding salt to a spray suspension reduces the recov-ery of airborne infectious Semliki Forest virus at high RH in acontrolled chamber (17). On the other hand, polyhydroxy com-pounds (17, 118) and peptones (69) are protective. Similarly,adding dextrose to the spray fluid significantly enhances therecovery of coliphage T3 at mid-range RH, but spermine, sper-mine-phosphate, thiourea, galacturonic acid, and glucosaminicacid have no effect on virus recovery (45). Mid-range RH andfecal matter as a spray fluid have been shown to enhance therecovery of a strain of human rotavirus (82). Organic matterand chemical compounds probably exert their protective effectby reducing desiccation and other environmental stresses.
Lastly, the gas composition of the air can also have aninfluence on viruses, as ozone has been shown to inactivateairborne viruses (96). In fact, virus susceptibility to ozone ismuch higher than those of bacterial and fungal bioaerosols(133). However, the ozone efficacy will vary from virus to virus.For example, phage �X174 is more susceptible to ozone thanare phages MS2 and T7 (133). Ions in the air can also reducethe recovery rate of certain viruses, such as aerosolized T1bacteriophage, with positive ions having the most detrimentaleffect (64).
AIR SAMPLING METHODOLOGIES
Most air sampling technologies depend on the aerodynamicdiameter of the airborne particles, the adhesion properties ofairborne particles, Brownian motion, thermal gradients, andthe inertia of the particles. Aerosolized particles attach to anysurface with which they come into contact (75). Adhesiveforces such as van der Waals forces, electrostatic forces, andsurface tension partly explain this adhesion. Most of the sam-pling methodologies presented in this review are based on thisprinciple.
Airborne particles with aerodynamic diameters on the orderof 100 nm or less are prone to a particular way of moving,mainly due to the billions of collisions they encounter with gasmolecules. This is called Brownian motion, and the smaller theparticle, the greater the movement and the more likely that the
particle will diffuse, come into contact with a surface, andadhere to it. When this happens, the other suspended particlesoccupy the space left vacant by the particle that has adhered tothe surface. This phenomenon is the basis for the efficientremoval of very small particles by filtration, particularly whenthe distance between two surfaces of the filter is sufficient forthe particles to pass through.
Larger particles with aerodynamic diameters on the order ofa micrometer or more are less influenced by Brownian motionbut have greater inertia. Gravitational attraction has a signif-icant impact on these particles and causes them to settle onsurfaces. These particles are also more easily diverted from agas streamline, leading to impaction on surfaces, especially athigh velocity and when the angle of the airflow is drasticallyaltered. Very small particles have less inertia and will morelikely follow the streamline.
AIR SAMPLING FOR VIRUS RECOVERY
Various sampling devices can be used to recover airborneviruses, and some are illustrated in Fig. 3. The most commonare liquid and solid impactors as well as filters. Electrostaticprecipitators have also been tested. Table 2 presents an exten-sive compilation of studies on the recovery of viral particles.The history of use of air samplers for viral aerosols is summa-rized in Fig. 1.
Solid Impactors
Solid impactors, such as Andersen samplers, slit samplers,and cyclone samplers, are usually more efficient at capturinglarge particles. Andersen and slit samplers accelerate the par-ticles through narrow holes or slits. The streamline movestoward a solid surface and abruptly changes direction. Theinertia of the particles deviates them from the airflow andimpacts them on the surface, which usually holds a petri dishwith a culture medium. The medium is either washed to collectthe particles or used directly for plaque assays. Andersen sam-plers contain a number of stages, each of which traps particlesof a specific aerodynamic size range, and is often used todetermine the sizes of virus-laden particles (54, 79, 132, 133).The multistage configuration is designed to accelerate the in-coming particles. The first stage induces moderate accelerationso that only the largest particles deviate from their trajectory.The second stage accelerates the smaller particles a little more,and so on. A six-stage Andersen sampler recovers particlesranging from 0.65 �m in diameter on the lowest stage to 7.5�m and over on the top stage. Single-stage Andersen samplerscan also be used to capture particles. The lower recovery limitof these samplers is a function of the diameter of the holesthrough which the particles are accelerated.
Slit samplers are used mostly to determine aerosol concen-trations of bacteria as a function of time. The acceleratedparticles are impacted onto a rotating petri dish containing aculture medium. This makes it possible to determine the timewhen each particle was sampled. Time-dependent results canbe obtained only if the samples are grown directly on the solidculture medium. Obviously, the use of a liquid medium orbuffer on top of a solid medium compromises this function.Nevertheless, slit samplers have been used with a liquid layer
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on the culture medium to recover viruses. The particles thatimpact the solid surface are immediately resuspended in aliquid medium to maximize the recovery of both infective vi-ruses and viral nucleic acids. This method was used successfullyin Toronto, Canada, during the 2003 SARS outbreak (19). Infact, two air sampling methods were used during this outbreak,namely, a modified high-resolution slit sampler system andpolytetrafluoroethylene (PTFE) membrane filters with 0.3-�mpores. The samples were tested for viruses by reverse tran-scriptase PCR (RT-PCR) and culture assays. The cultureswere all negative, and only 2 of the 10 slit samples were PCRpositive. These results might be explained by the absence or avery low concentration of airborne viruses.
Centrifugal forces have also been used to sample artificiallygenerated airborne influenza viruses. The samples collected bycentrifugation within an hour after aerosolization caused in-fluenza in inoculated ferrets (140). The centrifugal samplerrecovered close to 100% of 2.3-�m-diameter particles and 50%of 0.77-�m-diameter particles at 4,500 rpm, which correspondsto an airflow of 1.44 to 1.90 cubic feet/min (40 to 54 liters/min)(108). Errington and Powell developed small and large cycloneseparators. The small cyclone separator has a flow rate of 75liters/min, and the large one has a flow rate of 350 liters/min.Both cyclones accelerate the air by using a centrifugal vortex,pushing the airborne particles into contact with a solid surfaceby using the inertia of the particles. A scrubbing liquid isconstantly injected into the cyclone and collected in a bottle atits base. The concentration of the aerosol in the liquid dependson the air sampling and liquid injection rates. The smallersampler can, for example, concentrate 100 liters of air in 1 ml
of liquid (49). This first generation of cyclone separators in-spired the development of similar apparatuses, which can sam-ple the air at various rates.
A 170-liters/min flow rate (2- and 20-min samples) has beenused to sample air contaminated with FMD virus released byinfected pigs (7) as well as by sheep and heifers (8). In anotherstudy, a 300-liters/min flow rate (15-min sample) was success-fully used to sample the air of isolated units housing pigsinfected with Aujeszky’s disease virus. However, the samplerwas unable to detect low levels of airborne virus (20). Cyclonesamplers have also been used at high flow rates, ranging from700 to 1,000 liters/min (5- to 30-min samples), to recover air-borne viruses (38–40, 60, 62). A recirculating liquid cyclone-style air sampler operating at 265 liters/min (8-h sample), com-bined with culture and RT-PCR, has been used to detect exoticNewcastle disease virus in naturally contaminated commercialpoultry flocks (74). The capacity of cyclones to concentrateaerosols in large volumes of air over long, uninterrupted peri-ods is one of the major advantages of this type of sampler.However, some investigators have reported that cyclone sam-plers are much less efficient than other samplers at recoveringlow concentrations of airborne viruses (20). This may be due tothe physical stress caused by cyclone samplers, which maycause structural damage to the viruses and thus decrease theirinfectivity (20).
Liquid Impactors
All-glass impingers (AGIs) (Fig. 3), also called Porton im-pingers, and AGI-like samplers are the most often used sam-
FIG. 3. Diagrams of six different bioaerosol samplers. Red lines and arrows represent the airflow into the sampler. Blue arrows representairflow out of the sampler. These drawings are simplified representations.
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plers for the capture of airborne viruses (Fig. 1; Table 2). Theliquid impinger, which was first described by May and Harper(91), works by accelerating airborne particles through a narroworifice placed at a fixed distance from the bottom of a flaskcontaining a liquid. A pressure drop is created in the flask andforces the air to enter through the inlet of the impinger. Theair enters horizontally through a glass tube, which curves to avertical position, forcing the air to change direction and flowdownward. The diameter of the tubing abruptly narrows andacts as a critical flow orifice, accelerating the air passingthrough it to sonic velocity. The flow remains constant as longas there is at least half an atmosphere of suction in the im-pinger. The curve in the tube is intended to trap the largerparticles by inertial impaction and mimics the airway of thehuman nose. The largest particles entering the flask throughthe critical flow orifice are impacted onto the liquid. The for-mation of small bubbles in the liquid of the impinger can alsohelp to sample very small particles by diffusion. However, thereaerosolization of particles due to the scavenging propertiesof the air bubbles can be a problem, especially for hydrophobicparticles. The liquid prevents desiccation and facilitates theextraction of genetic material for subsequent analysis. TheAGI-4 sampler (the number refers to the distance, in millime-ters, separating the tip of the critical orifice from the bottom ofthe flask) and the AGI-30 sampler, also called a raised im-pinger, are often used as standard reference samplers (71).Multistage liquid impingers are also available. Particles im-pinge into liquids in successive stages as a function of theiraerodynamic size. This type of sampler, like the Andersensampler, is used mainly to determine the size distribution ofinfectious particles (39, 40).
According to Harstad (66), who compared the samplingefficiencies of two types of liquid impingers, two types of filters,and a fritted bubbler, using submicrometer aerosols of a sus-pension of bacteriophage T1 (a tailed bacterial virus) with aradioactive tracer, liquid impingers are the least destructivesamplers, with a relative efficiency, as determined by culture,superior by 18% to that of the next best sampler, although 30%to 48% of the sample was physically lost. Harstad also reportedthat filters are very destructive for this bacterial virus but arethe most efficient at collecting submicrometer particles andthat the fritted bubbler is the least efficient sampler, with aphysical loss of over 80% of the sample (66). These differencesin the recovery rates of AGI samplers and filters were con-firmed in a later study (64). The gentler sampling processleading to better recovery of infective viruses seems to be themain reason for the wide use of AGI samplers in aerovirology.Many studies involving airborne virus sampling have been con-ducted using the AGI-30 or AGI-4 sampler as the main sam-pling device (4, 10, 17, 42, 45–48, 57, 58, 65, 67, 78, 81, 82, 84,86, 105, 116, 118, 119, 122, 129–131, 138). Most were done todetermine the effects of various factors on the recovery rates ofairborne viruses. Although some studies indicate that the AGIhas a lower recovery potential than other samplers, such as thelarge-volume sampler (LVS) (94, 144), the Andersen sampler(127), and the slit sampler (126), other studies suggest that theAGI recovers concentrations of viruses that are equivalent tothose with the LVS (121), greater than those with theAndersen sampler (15), and greater than (30) or equal to (85)those with the slit sampler.
A recently developed impinger model, the “swirling aerosolcollector” (Fig. 3) (143), commercialized as the BioSampler,has also been used to study viral aerosols in the same way asAGIs (22, 72, 124). This newer impinger works much like theAGI, with a curved inlet tube and a vacuum in the flask to forcethe air through the sampler. The major difference is the num-ber and positions of nozzles. Instead of forcing air at sonicspeed through a single nozzle directed toward the base of theflask, as with the AGI, the BioSampler has three tangentialsonic nozzles. The collection liquid in the flask moves in aswirling motion during sampling. The sampling procedure isless violent and less destructive than that with the AGI-30sampler. Hermann et al. (73) studied the BioSampler andreported that it, as well as the AGI-30 sampler, collects signif-icantly more aerosolized porcine reproductive and respiratorysyndrome viruses than the AGI-4 sampler does. They alsoreported that the collection efficiency of the BioSampler issignificantly greater than that of the AGI-30 sampler after 15and 20 min of sampling (73). However, both the AGI-30 andthe BioSampler (as well as the frit bubbler) are surprisinglyinefficient at recovering submicrometer and ultrafine virusaerosols, with collection efficiencies of �10% for all threesamplers for the 30- to 100-nm particle size range (76).
Prehumidifying aerosols by using a humidifier bulb in com-bination with an AGI-30 sampler can have both positive andnegative effects on the recovery of infectious viruses from air-borne material, depending on the virus. The AGI-30 with hu-midifier bulb has been shown to increase the recovery of air-borne coliphages T3 (67, 137), T2, and T7 (16), as well asPasteurella pestis bacteriophages (67). Recovery increases five-fold at high RH and up to 1,000-fold at low RH when apeptone solution or saliva is used as the spray medium. How-ever, adding NaCl to the spray medium has no effect on therecovery of T3 (131). Prehumidification has no effect on therecovery of mengovirus 37A (Picornaviridae family; ssRNAvirus) or vesicular stomatitis virus (Rhabdoviridae family;ssRNA virus) but increases the recovery of bacteriophage S13(Microviridae family; ssDNA virus) at mid-range RH (137).One possible explanation for the beneficial effect of prehu-midification may be that the median size of the particles isincreased at high RH by the condensation of the water vaporon the airborne particles. The condensation may also have anegative effect by dissolving the particle nuclei, exposing theviruses to high concentrations of solutes, which may structur-ally damage the virus, leading to a loss of infectivity. No de-finitive explanation has yet been proposed to explain the effectof prehumidification on viral infectivity.
Filters
Since most samplers cannot efficiently trap particles with anaerodynamic size of �500 nm, filters are frequently used tosample airborne viruses. Filter efficiency is based on the fol-lowing five basic mechanisms: (i) interception, (ii) inertial im-paction, (iii) diffusion, (iv) gravitational settling, and (v) elec-trostatic attraction (75). While each mechanism depends onthe aerodynamic diameter of the particle, interception alsodepends on particle radius. Interception occurs when a particlefollows the streamline going around an obstacle but, becauseof its size, comes into contact with and is intercepted by the
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 419
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Dow
nloaded from
TA
BL
E2.
Sum
mar
yof
viru
sae
roso
lsam
plin
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s
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
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plin
gen
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nmen
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irus
and/
ortr
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icle
size
(�m
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naly
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Com
men
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olso
urce
Ref
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icat
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fluen
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stra
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o8
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ulat
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offe
rret
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sin
ocul
ated
with
mat
eria
lco
llect
edfr
omth
eai
rco
ntra
cted
influ
enza
whe
nth
esa
mpl
ing
was
done
with
inan
hour
afte
rth
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ater
ialw
assu
spen
ded
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miz
er14
019
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plin
gat
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ers
540
and
1,08
0lit
ers
Poul
try
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oenc
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ocul
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nof
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suffi
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tvi
ruse
sto
infe
ctch
ick
embr
yos
Poul
try
3519
48
Impi
nger
s11
liter
s/m
info
r15
to12
0s
Mod
ified
Hen
ders
onap
para
tus
and
rota
ting
stai
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sst
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rum
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cini
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rus,
influ
enza
Avi
rus
stra
inPR
8,V
enez
uela
neq
uine
ence
phal
omye
litis
(VE
E)
viru
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liom
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I(B
runh
ilde)
,fo
rmal
in-k
illed
Pas
teur
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tula
rens
isce
llsla
bele
dw
ith32P
for
VE
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rus
and
viru
ssu
spen
sion
labe
led
with
32P
for
the
othe
rth
ree
viru
ses
Inoc
ulat
ion
ofeg
gsor
mic
e,cu
lture
Air
born
evi
ruse
sw
ere
reco
vere
dup
to23
haf
ter
aero
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nat
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rent
RH
and
tem
pera
ture
s
Col
lison
atom
izer
6519
61
Slit
sam
pler
(sol
idge
latin
med
ium
)1
ft3/m
info
r1
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500-
liter
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igla
sch
ambe
rB
acte
riop
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T3
0.5–
1(9
6%of
part
icle
s)C
ultu
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itsa
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ith12
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latin
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ium
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vere
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75%
ofph
ages
com
pare
dto
AG
I-30
sam
pler
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onef
rin
nebu
lizer
3019
61
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I-30
12.8
liter
s/m
info
r15
min
1,50
0-lit
erPl
exig
las
cham
ber
Bac
teri
opha
geT
30.
5–1
(96%
ofpa
rtic
les)
Cul
ture
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sam
pler
with
a12
%ge
latin
med
ium
reco
vere
d�
75%
ofph
ages
com
pare
dto
AG
I-30
sam
pler
Vap
onef
rin
nebu
lizer
3019
61
Slit
sam
pler
1ft
3/m
info
r4
and
15m
inM
odifi
edH
ende
rson
appa
ratu
sV
EE
viru
sIn
ocul
atio
nof
mic
e,tit
ratio
nw
ithem
bryo
nate
dhe
n’s
eggs
,and
cultu
re
Res
ults
with
the
slit
sam
pler
and
the
AG
I-30
sam
pler
wer
eco
mpa
rabl
e
Col
lison
gene
rato
r85
1961
AG
I-30
12.5
liter
s/m
info
r4
and
15m
inM
odifi
edH
ende
rson
appa
ratu
sV
EE
viru
sIn
ocul
atio
nof
mic
e,tit
ratio
nw
ithem
bryo
nate
dhe
n’s
eggs
,and
cultu
re
Res
ults
with
the
slit
sam
pler
and
the
AG
I-30
sam
pler
wer
eco
mpa
rabl
e
Col
lison
gene
rato
r85
1961
Port
onim
ping
er10
to20
liter
s/m
info
r0.
5an
d1
min
14-f
t3ae
roso
lch
ambe
rw
ithfa
nB
acte
riop
hage
T3
Cul
ture
The
elec
tros
tatic
prec
ipita
tor
was
very
effic
ient
;ozo
neca
nbe
prod
uced
athi
ghR
Hw
ithth
eel
ectr
osta
ticpr
ecip
itato
r;a
0.1%
pept
one
solu
tion
can
prot
ect
phag
eT
3fr
omoz
one
Col
lison
spra
y99
1961
420 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
on May 8, 2020 by guest
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Dow
nloaded from
Ele
ctro
stat
icpr
ecip
itato
r5
to40
liter
s/m
info
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min
14-f
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roso
lch
ambe
rw
ithfa
nB
acte
riop
hage
T3
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ture
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elec
tros
tatic
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ipita
tor
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very
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ient
;ozo
neca
nbe
prod
uced
athi
ghR
Hw
ithth
eel
ectr
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ticpr
ecip
itato
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sssa
mpl
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ntai
ning
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cked
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cott
on
20to
18,0
00lit
ers
Hos
pita
lV
ario
lavi
rus
Inoc
ulat
ion
ofch
ick
embr
yos
Vir
uses
wer
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cove
red
in1
of38
sam
ples
Hum
ans
9519
61
Mod
ified
capi
llary
impi
nger
s11
liter
s/m
info
r10
s4-
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cond
ition
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riop
hage
T5,
influ
enza
viru
s,po
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isvi
rus
5–6
(mea
nsi
ze5
cmfr
omth
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tlet)
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ture
Rec
over
yde
pend
edon
the
aero
soliz
atio
nm
ediu
man
dth
eR
H,
but
the
effe
ctof
RH
did
not
depe
ndon
the
com
posi
tion
ofth
em
ediu
m;t
hesu
rviv
alcu
rves
atdi
ffere
ntR
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ere
not
the
sam
efo
rin
fluen
zaan
dpo
liom
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isvi
ruse
s
All-
glas
sin
dire
ct-
type
spra
y69
1962
AG
I-30
12.5
liter
s/m
info
r1
min
1,60
0-lit
erPl
exig
las
cham
ber
with
fan
Bac
teri
opha
geT
30.
5–5.
0(p
eak
at2.
0�
m)
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ture
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best
reco
very
was
athi
ghR
HH
artm
anat
omiz
er45
1964
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t-st
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GI
12.0
liter
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info
r5
to20
min
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pita
lroo
ms
ofin
fect
edpa
tient
sV
irus
esas
soci
ated
with
resp
irat
ory
dise
ases
Cul
ture
Vir
uses
wer
ere
cove
red
in1
of23
tria
lsH
uman
s10
1964
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uid
impi
nger
sA
GI-
412
.5lit
ers/
min
for
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eros
olch
ambe
rB
acte
riop
hage
T1,
32P
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sm
edia
ndi
amet
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0.2
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ture
Rec
over
edth
em
ost
viab
leph
ages
,but
maj
orsa
mpl
elo
ss(3
0to
40%
)
Dau
treb
and
D30
1ae
roso
lgen
erat
or66
1965
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illar
yim
ping
er2.
5L
/min
for
5m
inA
eros
olch
ambe
rB
acte
riop
hage
T1,
32P
Mas
sm
edia
ndi
amet
er,
0.2
Cul
ture
Rec
over
edth
em
ost
viab
leph
ages
,but
maj
orsa
mpl
elo
ss(3
0to
40%
)
Dau
treb
and
D30
1ae
roso
lgen
erat
or66
1965
Filt
ers
Che
mic
alC
orps
type
61
liter
/min
(8cm
/s)
for
5m
inA
eros
olch
ambe
rB
acte
riop
hage
T1,
32P
Mas
sm
edia
ndi
amet
er,
0.2
Cul
ture
Ver
yde
stru
ctiv
ebu
tm
ost
effic
ient
colle
ctio
nof
subm
icro
met
erai
rbor
nepa
rtic
les
Dau
treb
and
D30
1ae
roso
lgen
erat
or66
1965
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ssfil
ter
pape
r(M
SA11
06B
H)
1lit
er/m
in(8
cm/s
)fo
r5
min
Aer
osol
cham
ber
Bac
teri
opha
geT
1,32P
Mas
sm
edia
ndi
amet
er,
0.2
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ture
Ver
yde
stru
ctiv
ebu
tm
ost
effic
ient
colle
ctio
nof
subm
icro
met
erai
rbor
nepa
rtic
les
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treb
and
D30
1ae
roso
lgen
erat
or66
1965
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tted
bubb
ler
1lit
er/m
info
r5
min
Aer
osol
cham
ber
Bac
teri
opha
geT
1,32P
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sm
edia
ndi
amet
er,
0.2
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ture
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rtan
tsa
mpl
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ss(�
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)D
autr
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dD
301
aero
solg
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ator
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onim
ping
er10
liter
s/m
info
r5
to10
min
Hos
pita
lroo
ms
Smal
lpox
viru
sIn
ocul
atio
nof
chic
kem
bryo
sL
arge
part
icle
sfr
ompa
tient
s’be
dcl
othe
sse
emed
tobe
mos
tlyre
spon
sibl
efo
rco
ntam
inat
ion
ofth
eai
rin
the
vici
nity
ofpa
tient
s
Hum
ans
4119
65
Con
tinue
don
follo
win
gpa
ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 421
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
Sam
plin
gen
viro
nmen
tV
irus
and/
ortr
acer
Part
icle
size
(�m
)A
naly
tical
met
hod
Com
men
tsA
eros
olso
urce
Ref
eren
ceY
rof
publ
icat
ion
Petr
idis
h(s
ettli
ngpl
ates
)5
to10
min
Hos
pita
lroo
ms
Smal
lpox
viru
sIn
ocul
atio
nof
chic
kem
bryo
sL
arge
part
icle
sfr
ompa
tient
s’be
dcl
othe
sse
emed
tobe
mos
tlyre
spon
sibl
efo
rco
ntam
inat
ion
ofth
eai
rin
the
vici
nity
ofpa
tient
s
Hum
ans
4119
65
Top
part
ofan
And
erse
nsa
mpl
er
10lit
ers/
min
for
5to
10m
inH
ospi
talr
oom
sSm
allp
oxvi
rus
Inoc
ulat
ion
ofch
ick
embr
yos
Lar
gepa
rtic
les
from
patie
nts’
bed
clot
hes
seem
edto
bem
ostly
resp
onsi
ble
for
cont
amin
atio
nof
the
air
inth
evi
cini
tyof
patie
nts
Hum
ans
4119
65
Cap
illar
yim
ping
ers
4-m
3co
nditi
oned
room
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sles
viru
s5
(ave
rage
diam
eter
)C
ultu
reR
ecov
ery
depe
nded
onR
H34
1965
LV
S1,
785
ft3
for
5m
in1,
440-
ft3
Arm
yho
spita
lroo
mA
deno
viru
sty
pe4
Cul
ture
Via
ble
viru
ses
wer
ere
cove
red
atve
rylo
wco
ncen
trat
ions
Hum
ans
1119
66
LV
S10
,000
liter
s/m
info
r3.
5m
in32
,800
-lite
rro
omw
ithat
omiz
edvi
rus
susp
ensi
on
Cox
sack
ievi
rus
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pe21
,sod
ium
fluor
esce
in
1–15
Cul
ture
LV
Sco
nsis
tent
lyre
cove
red
mor
eflu
ores
cein
than
the
AG
I-30
did
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vers
ityof
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cago
Tox
icity
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orat
orie
sat
omiz
er
5419
66
AG
I-30
12.5
liter
s/m
info
r1
min
32,8
00-li
ter
room
with
atom
ized
viru
ssu
spen
sion
Cox
sack
ievi
rus
Aty
pe21
,sod
ium
fluor
esce
in
1–15
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ture
LV
Sco
nsis
tent
lyre
cove
red
mor
eflu
ores
cein
than
the
AG
I-30
did
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vers
ityof
Chi
cago
Tox
icity
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orat
orie
sat
omiz
er
5419
66
Ele
ctro
stat
icpr
ecip
itato
rR
oom
sco
ntai
ning
infe
cted
rabb
itsR
abbi
tpo
xvi
rus
stra
inU
trec
htan
dR
ocke
felle
rIn
stitu
test
rain
Cul
ture
Low
conc
entr
atio
nsw
ere
reco
vere
dw
ithth
eel
ectr
osta
ticpr
ecip
itato
r,an
dno
new
asre
cove
red
with
the
impi
nger
Rab
bits
141
1966
Rai
sed
glas
sim
ping
erR
oom
sco
ntai
ning
infe
cted
rabb
itsR
abbi
tpo
xvi
rus
stra
inU
trec
htan
dR
ocke
felle
rIn
stitu
test
rain
Cul
ture
Low
conc
entr
atio
nsw
ere
reco
vere
dw
ithth
eel
ectr
osta
ticpr
ecip
itato
r,an
dno
new
asre
cove
red
with
the
impi
nger
Rab
bits
141
1966
AG
I-4
12.5
liter
s/m
info
r5
min
Aer
osol
cham
ber
Bac
teri
opha
geT
1�
1C
ultu
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heA
GI-
4sa
mpl
erre
cove
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mor
eai
rbor
nevi
ruse
sth
anty
pe6
filte
rpa
per
did;
ions
affe
cted
the
stab
ility
ofsu
bmic
rom
eter
T1
phag
e
Dau
treb
ande
aero
sol
gene
rato
r64
1966
Che
mic
alC
orps
type
6fil
ter
pape
r
5m
inat
1.0
liter
/m
inA
eros
olch
ambe
rB
acte
riop
hage
T1
�1
Cul
ture
The
AG
I-4
sam
pler
reco
vere
dm
ore
airb
orne
viru
ses
than
type
6fil
ter
pape
rdi
d;io
nsaf
fect
edth
est
abili
tyof
subm
icro
met
erT
1ph
age
Dau
treb
ande
aero
sol
gene
rato
r64
1966
AG
I-30
12.5
liter
s/m
info
r5
min
Rot
atin
gdr
umC
olum
bia
SKgr
oup
viru
ses
Cul
ture
Inac
tivat
ion
ofth
eai
rbor
nevi
ruse
sde
pend
edon
RH
Mod
ified
Wel
lsre
fluxi
ngat
omiz
er4
1966
AG
I12
liter
s/m
in(3
-lite
rsa
mpl
es)
140-
liter
alum
inum
drum
New
cast
ledi
seas
evi
rus,
infe
ctio
usbo
vine
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otra
chei
tisvi
rus,
vesi
cula
rst
omat
itis
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s,T
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age,
rhod
amin
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ture
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tre
cove
ryat
low
RH
for
New
cast
ledi
seas
evi
rus
and
vesi
cula
rst
omat
itis
viru
san
dat
high
RH
for
bovi
nerh
inot
rach
eitis
viru
san
dT
3
De
Vilb
iss
no.4
0ne
buliz
er12
219
67
LV
S10
,000
liter
s/m
info
r6
or7
min
1,44
0-ft
3A
rmy
hosp
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oom
Ade
novi
rus
Cul
ture
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luni
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ples
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67
422 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
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for
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imal
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ples
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odel
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als
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ples
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419
68
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nger
2m
in50
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erro
tatin
gto
roid
drum
Men
govi
rus
37A
Cul
ture
Inac
tivat
ion
ofth
evi
rus
was
due
toda
mag
eto
the
viri
onst
ruct
ure
Mod
ified
Wel
lsre
fluxi
ngat
omiz
er5
1968
LV
Sw
ithad
ded
prei
mpa
ctor
357
ft3
for
1m
inor
full
room
for
3m
in
986-
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onin
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ects
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over
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air
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ples
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ectio
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rtic
les
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both
smal
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part
icle
aero
sols
Hum
ans
1219
68
LV
Sm
odel
M1,
000
liter
s/m
info
r1
h3.
65-
by3.
35-
by3.
05-m
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Fou
rst
rain
sof
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rus
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Lom
bard
y,01
Swis
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1B
FS
1860
,and
02B
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ia)
Cul
ture
and
inoc
ulat
ion
ofun
wea
ned
mic
e
The
amt
ofvi
rus
reco
vere
dby
the
impi
nger
was
sim
ilar
toth
atre
cove
red
byth
eL
VS
Cat
tle,s
heep
,and
pigs
121
1969
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tista
geliq
uid
impi
nger
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rst
rain
sof
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bard
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FS
1860
,and
02B
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ia)
Cul
ture
and
inoc
ulat
ion
ofun
wea
ned
mic
e
The
amt
ofvi
rus
reco
vere
dby
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impi
nger
was
sim
ilar
toth
atre
cove
red
byth
eL
VS
Cat
tle,s
heep
,and
pigs
121
1969
AG
I-30
Dua
l-aer
osol
tran
spor
tap
para
tus
T3
and
S13
colip
hage
s,m
engo
viru
s-37
A,
and
vesi
cula
rst
omat
itis
viru
s
Cul
ture
Whi
leth
ere
cove
ryof
som
evi
ruse
sfr
omae
roso
lsw
asin
crea
sed
bypr
ehum
idifi
catio
n,no
gene
raliz
atio
nco
uld
bedr
awn
Mod
ified
Wel
lsre
flux
atom
izer
137
1969
AG
I-30
with
ahu
mid
ifier
bulb
Dua
l-aer
osol
tran
spor
tap
para
tus
T3
and
S13
colip
hage
s,m
engo
viru
s-37
A,
and
vesi
cula
rst
omat
itis
viru
s
Cul
ture
Whi
leth
ere
cove
ryof
som
evi
ruse
sfr
omae
roso
lsw
asin
crea
sed
bypr
ehum
idifi
catio
n,no
gene
raliz
atio
nco
uld
bedr
awn
Mod
ified
Wel
lsre
flux
atom
izer
137
1969
AG
I-30
12.5
liter
s/m
inT
wo
500-
liter
rota
ting
drum
sB
acte
riop
hage
T3
Cul
ture
Vir
usre
cove
ryw
asbe
stat
high
erR
HA
tom
izer
desi
gned
byth
ela
b(s
imila
rto
the
Wel
lsat
omiz
er)
138
1969
Rai
sed
impi
nger
sSe
mlik
iFor
est
viru
s,w
ashe
dB
acill
ussu
btili
ssp
ores
Cul
ture
Bes
tre
cove
ryat
low
RH
,w
ithgo
odpr
otec
tive
effe
ctof
poly
hydr
oxy
com
poun
dsat
low
RH
Col
lison
spra
y17
1969
AG
I-30
,with
orw
ithou
ta
hum
idifi
erbu
lb
Tw
o50
0-lit
erro
tatin
gdr
ums
Bac
teri
opha
geT
3an
dP
aste
urel
lape
stis
bact
erio
phag
e
1–5
Cul
ture
Preh
umid
ifica
tion
ofth
eai
rsa
mpl
essi
gnifi
cant
lyen
hanc
edth
ere
cove
ryof
airb
orne
viru
ses
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ilar
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ells
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)
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teri
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ture
RH
and
aero
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nha
da
maj
orim
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ralr
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Mod
ified
Wel
lsre
flux
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I-30
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lb
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teri
opha
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ralr
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70
Con
tinue
don
follo
win
gpa
ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 423
on May 8, 2020 by guest
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Dow
nloaded from
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BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
Sam
plin
gen
viro
nmen
tV
irus
and/
ortr
acer
Part
icle
size
(�m
)A
naly
tical
met
hod
Com
men
tsA
eros
olso
urce
Ref
eren
ceY
rof
publ
icat
ion
Slit
sam
pler
with
adhe
sive
surf
ace
petr
idi
shes
Roo
mco
ntai
ning
infe
cted
rabb
its(4
,500
ft3)
Rab
bit
poxv
irus
stra
inU
trec
htC
ultu
reM
ore
viru
ses
wer
ere
cove
red
inth
eto
pst
ages
ofth
eA
nder
sen
sam
pler
than
inth
elo
wer
stag
es;b
oth
the
slit
sam
pler
san
dth
eA
nder
sen
sam
pler
succ
essf
ully
reco
vere
dai
rbor
nevi
ruse
s
Rab
bits
128
1970
Aut
omat
edsl
itsa
mpl
erw
ithad
hesi
vesu
rfac
epe
tri
dish
es
1h
(60
ft3)
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mco
ntai
ning
infe
cted
rabb
its(4
,500
ft3)
Rab
bit
poxv
irus
stra
inU
trec
htC
ultu
reM
ore
viru
ses
wer
ere
cove
red
inth
eto
pst
ages
ofth
eA
nder
sen
sam
pler
than
inth
elo
wer
stag
es;b
oth
the
slit
sam
pler
san
dth
eA
nder
sen
sam
pler
succ
essf
ully
reco
vere
dai
rbor
nevi
ruse
s
Rab
bits
128
1970
And
erse
nsa
mpl
erw
ithad
hesi
vesu
rfac
epe
tri
dish
es
1h
(60
to12
0ft
3)
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mco
ntai
ning
infe
cted
rabb
its(4
,500
ft3)
Rab
bit
poxv
irus
stra
inU
trec
htC
ultu
reM
ore
viru
ses
wer
ere
cove
red
inth
eto
pst
ages
ofth
eA
nder
sen
sam
pler
than
inth
elo
wer
stag
es;b
oth
the
slit
sam
pler
san
dth
eA
nder
sen
sam
pler
succ
essf
ully
reco
vere
dai
rbor
nevi
ruse
s
Rab
bits
128
1970
Mod
ified
And
erse
nsa
mpl
er1
ft3/m
inA
eros
olap
para
tus
(Hen
ders
on)
Vac
cini
avi
rus,
polio
viru
sC
ultu
reT
hem
odifi
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nder
sen
sam
pler
gave
the
best
perc
enta
gere
cove
ry,f
ollo
wed
byth
eim
ping
eran
dth
esl
itsa
mpl
er;
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adhe
sive
surf
ace
sam
plin
gm
etho
din
dica
ted
the
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ofvi
rus-
bear
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part
icle
s
Spra
y12
719
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sam
pler
with
sucr
ose,
glyc
erol
,and
bovi
nese
rum
albu
min
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B)
med
ium
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info
r0.
5to
10m
inA
eros
olap
para
tus
(Hen
ders
on)
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cini
avi
rus,
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viru
sC
ultu
reT
hem
odifi
edA
nder
sen
sam
pler
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cove
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ollo
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er;
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plin
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nsa
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the
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ead
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hod
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anga
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rus,
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Salts
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eae
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ere
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viru
s
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424 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
on May 8, 2020 by guest
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Poul
try
orC
ollis
onat
omiz
er77
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cade
impa
ctor
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ers/
min
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10-f
tby
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oms,
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try
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ithin
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edch
icke
ns,
and
am
odifi
edH
ende
rson
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sw
itha
500-
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rota
ting
stai
nles
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um
Thr
eest
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seas
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vere
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ithal
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ition
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ollis
onat
omiz
er77
1973
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impi
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min
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10-f
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10-f
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oms,
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ithin
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gsV
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vere
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ithal
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ffere
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ing
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ition
s
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try
orC
ollis
onat
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er77
1973
AG
I-30
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in50
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tatin
gdr
umSi
mia
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rus
402
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ndi
amet
er)
Cul
ture
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wer
est
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atal
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sted
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ere
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-ran
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er6
1973
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posi
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ium
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very
Dir
ect
spra
yap
para
tus
(FK
8)13
019
73
Con
tinue
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follo
win
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ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 425
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
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Sam
pler
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plin
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gen
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icle
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icat
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nded
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ivity
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aine
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ged
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8di
rect
-typ
ene
buliz
er32
1973
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tista
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ase
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irus
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vere
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omst
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ofth
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120
1974
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ultis
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er27
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uses
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ctiv
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ium
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r30
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lture
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ates
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ples
wer
eal
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ativ
e
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ans
126
1974
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er11
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ards
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ates
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ples
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eal
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ans
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ates
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ples
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ans
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info
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ater
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ies
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ield
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ater
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ater
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the
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ialy
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cted
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stre
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oir,
nebu
lizer
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hum
ans
107
1976
426 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
on May 8, 2020 by guest
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sean
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odam
ine
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red
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rent
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nded
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1979
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ans
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1979
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r1
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atic
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ber
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ian
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ture
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viru
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rtic
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hica
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119
82
Con
tinue
don
follo
win
gpa
ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 427
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
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plin
gen
viro
nmen
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irus
and/
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acer
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icle
size
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5-�
mpo
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ze)
2.0
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info
r�
6h,
�18
h,or
24h
Hos
pita
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ms
Res
pira
tory
sync
ytia
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SV)
PCR
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edde
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met
hods
RSV
DN
Aw
asde
tect
edin
17of
the
27ro
oms
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ing
infe
cted
patie
nts
and
in32
ofth
e14
3sa
mpl
es
Hum
ans
319
98
Surf
ace
air
syst
emag
ariz
edte
rrai
nim
pact
or
1,80
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ers
indo
ors
and
3,00
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ers
outd
oors
Urb
anse
wag
epl
ants
Reo
viru
san
den
tero
viru
sC
ultu
reB
oth
viru
ses
wer
ede
tect
edin
som
esa
mpl
esU
rban
sew
age
trea
tmen
tpl
ants
2520
00
AG
I-30
28-m
3is
olat
ion
room
sA
ujes
zky’
sdi
seas
evi
rus
Cul
ture
No
viru
sw
asde
tect
edPi
gs58
2000
AG
I-30
12.5
liter
s/m
info
r10
min
Exh
aust
air
from
anin
fect
edba
rnPo
rcin
ere
prod
uctiv
ean
dre
spir
ator
ysy
ndro
me
viru
s(P
RR
SV)
PCR
and
cultu
reN
ovi
rus
was
dete
cted
inth
eai
rsa
mpl
esby
PCR
orby
cultu
re
Pigs
105
2002
All-
glas
scy
clon
esa
mpl
er17
0lit
ers/
min
for
20m
inA
nim
alro
oms
FM
Dvi
rus
OU
KG
34/2
001
Cul
ture
Vir
uses
wer
ere
cove
red
with
both
sam
pler
sSh
eep
and
heife
rs8
2002
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ee-s
tage
liqui
dim
ping
er55
liter
s/m
info
r5
min
610-
liter
cabi
net
FM
Dvi
rus
OU
KG
34/2
001
Cul
ture
Vir
uses
wer
ere
cove
red
with
both
sam
pler
sSh
eep
and
heife
rs8
2002
All-
glas
scy
clon
esa
mpl
er17
0lit
ers/
min
for
2m
inC
ham
ber
FM
Dvi
rus
stra
inO
1L
ausa
nne
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5C
ultu
reF
MD
viru
sw
asre
cove
red
from
the
air
sam
ples
,but
the
effic
ienc
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the
sam
pler
sw
asno
tco
mpa
red
Pigs
720
02
Con
tinue
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ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 431
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
Sam
plin
gen
viro
nmen
tV
irus
and/
ortr
acer
Part
icle
size
(�m
)A
naly
tical
met
hod
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men
tsA
eros
olso
urce
Ref
eren
ceY
rof
publ
icat
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Port
onA
GI
10to
13lit
ers/
min
for
2or
5m
inC
ham
ber
FM
Dvi
rus
stra
inO
1L
ausa
nne
Sw/6
5C
ultu
reF
MD
viru
sw
asre
cove
red
from
the
air
sam
ples
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tth
eef
ficie
ncy
ofth
esa
mpl
ers
was
not
com
pare
d
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720
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Thr
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tage
liqui
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liter
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info
r5
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mbe
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sst
rain
O1
Lau
sann
eSw
/65
Cul
ture
FM
Dvi
rus
was
reco
vere
dfr
omth
eai
rsa
mpl
es,
but
the
effic
ienc
yof
the
sam
pler
sw
asno
tco
mpa
red
Pigs
720
02
Mod
ified
SAS-
100
100
liter
s/m
info
r2.
5m
inW
ithin
and
arou
ndpo
ultr
ybr
oile
rho
uses
Mal
e-sp
ecifi
cco
lipha
ges
Cul
ture
Col
ipha
ges
wer
ere
cove
red
from
air
sam
ples
whe
na
prem
oist
ened
cellu
lose
este
rfil
ter
colle
ctio
nm
ediu
mw
asus
edfo
rsa
mpl
ing
byim
pact
ion
Poul
try
5020
02
PTF
Efil
ters
(2.0
-�m
pore
size
)10
min
Cha
mbe
rw
ithU
Vlig
htR
hino
viru
s16
stra
in11
757
from
AT
CC
,ur
anin
e
2(m
ass-
med
ian
diam
eter
ofdr
ople
ts)
Sem
ines
ted
RT
-PC
RT
hede
tect
ion
limit
was
1.3
50%
tissu
ecu
lture
infe
ctiv
edo
ses/
filte
rfo
rae
roso
lized
viru
s
Six-
jet
Col
lison
nebu
lizer
(CN
-38)
101
2003
MD
8ai
rsa
mpl
erw
ithst
erile
gela
tinm
embr
ane
filte
r(3
-�m
pore
size
)
100
liter
sin
1or
2m
inD
airy
fact
ory,
clos
epr
oxim
ityto
aru
nnin
gw
hey
sepa
rato
r
Lac
toco
ccus
lact
isba
cter
ioph
ages
Cul
ture
The
MD
8an
dA
irPo
rtM
D8
resu
ltsw
ere
very
sim
ilar;
the
phag
ere
cove
ryra
tes
for
the
MA
S-10
0(i
mpa
ctio
n)sa
mpl
erw
ere
1to
5%th
atfo
rth
eM
D8
(filtr
atio
n)sa
mpl
er
Whe
yse
para
tor
ina
dair
yfa
ctor
y10
320
03
Air
Port
MD
8sa
mpl
erw
ithst
erile
gela
tinm
embr
ane
filte
rs(3
-�m
pore
size
)
100
liter
sin
2m
inD
airy
fact
ory,
clos
epr
oxim
ityto
aru
nnin
gw
hey
sepa
rato
r
Lac
toco
ccus
lact
isba
cter
ioph
ages
Cul
ture
The
MD
8an
dA
irPo
rtM
D8
resu
ltsw
ere
very
sim
ilar;
the
phag
ere
cove
ryra
tes
for
the
MA
S-10
0(i
mpa
ctio
n)sa
mpl
erw
ere
1to
5%th
atfo
rth
eM
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(filtr
atio
n)sa
mpl
er
Whe
yse
para
tor
ina
dair
yfa
ctor
y10
320
03
MA
S-10
0de
vice
(with
five
diffe
rent
setu
ps)
100
liter
sin
1m
inD
airy
fact
ory,
clos
epr
oxim
ityto
aru
nnin
gw
hey
sepa
rato
r
Lac
toco
ccus
lact
isba
cter
ioph
ages
Cul
ture
The
MD
8an
dA
irPo
rtM
D8
resu
ltsw
ere
very
sim
ilar;
the
phag
ere
cove
ryra
tes
for
the
MA
S-10
0(i
mpa
ctio
n)sa
mpl
erw
ere
1to
5%th
atfo
rth
eM
D8
(filtr
atio
n)sa
mpl
er
Whe
yse
para
tor
ina
dair
yfa
ctor
y10
320
03
AG
I-30
12.5
liter
s/m
info
r10
min
Exh
aust
air
from
anin
fect
edba
rnPR
RSV
PCR
,cul
ture
,and
pig
bioa
ssay
All
168
air
sam
ples
wer
ene
gativ
eby
PCR
,cul
ture
,an
dpi
gbi
oass
ay
Pigs
129
2004
PTF
Efil
ters
(2.0
-�m
pore
size
)A
vera
geof
47h,
from
9a.
m.t
o5
p.m
.at
4lit
ers/
min
Offi
cebu
ildin
gsPi
corn
avir
uses
(rhi
novi
rus
and
ente
rovi
ruse
s)
Nes
ted
RT
-PC
Ran
dse
quen
cing
Fift
y-ei
ght
(32%
)of
181
filte
rsw
ere
posi
tive
for
pico
rnav
irus
Hum
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102
2004
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blin
gsa
mpl
er4
liter
s/m
info
r5
min
400-
liter
aero
sol
cham
ber
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enza
viru
sA
/Aic
hi/
2/68
(H3N
2),v
acci
nia
viru
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rain
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P(C
0355
K06
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uran
ine
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ority
ofpa
rtic
les)
Cul
ture
and
titra
tion
onch
icke
nem
bryo
s
The
aver
age
reco
very
rate
was
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for
the
influ
enza
viru
san
d89
%fo
rth
eva
ccin
iavi
rus
Col
lison
nebu
lizer
220
05
432 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
Port
able
,sin
gle-
siev
e,M
icro
Bio
MB
1im
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or
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to60
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estic
toile
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teri
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plin
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heim
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orsa
mpl
erre
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ling
plat
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estic
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omB
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hage
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ture
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teri
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ere
cove
red
with
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orsa
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red
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r18
min
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plin
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ads
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ato
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in)
Hos
pita
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ms
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ithSA
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rona
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ere
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posi
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rene
gativ
e;th
ePT
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mem
bran
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ters
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inth
eot
her
room
sw
ere
allP
CR
and
cultu
rene
gativ
e;lo
wco
ncen
trat
ions
(or
abse
nce)
ofai
rbor
nevi
ruse
sm
ayex
plai
nth
ene
gativ
ere
sults
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ans
1920
05
PTF
Em
embr
ane
filte
rs(0
.3-�
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resi
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min
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to13
hH
ospi
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sof
patie
nts
with
SAR
SSA
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coro
navi
rus
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-PC
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quan
titat
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lture
,and
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Ase
quen
cing
Tw
oof
10sa
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esfr
omth
ero
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are
cove
ring
SAR
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cted
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gth
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erw
ere
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posi
tive
but
cultu
rene
gativ
e;th
ePT
FE
mem
bran
efil
ters
used
inth
eot
her
room
sw
ere
allP
CR
and
cultu
rene
gativ
e;lo
wco
ncen
trat
ions
(or
abse
nce)
ofai
rbor
nevi
ruse
sm
ayex
plai
nth
ene
gativ
ere
sults
Hum
ans
1920
05
Port
able
air
sam
pler
450
liter
s/m
in10
.16-
cm-d
iam
eter
poly
viny
lchl
orid
epi
peof
3to
150
mlo
ngat
tach
edto
abl
ower
PRR
SVst
rain
MN
30-
100
Qua
ntita
tive
PCR
and
cultu
reV
irus
esw
ere
reco
vere
dfr
oma
dist
ance
ofup
to15
0m
Coo
king
oils
pritz
er31
2005
Cyc
lone
sam
pler
s70
0(
50)
liter
s/m
info
r5
min
Cha
mbe
rB
acte
riop
hage
MS2
Cul
ture
Vir
uses
wer
ere
cove
red
One
ortw
oth
ree-
jet
orsi
x-je
tC
ollis
onne
buliz
ers
6220
05
Wet
ted-
wal
lcyc
lone
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yle
air
sam
pler
265
liter
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info
r8
hC
omm
erci
alpo
ultr
yflo
cks
Exo
ticN
ewca
stle
dise
ase
viru
sR
eal-t
ime
RT
-PC
R(R
RT
-PC
R),
inoc
ulat
ion
ofeg
gs,c
ultu
re,a
ndse
quen
cing
Low
conc
entr
atio
nsof
viru
spa
rtic
les
wer
ede
tect
edby
RR
T-P
CR
;cu
lture
was
appa
rent
lym
ore
sens
itive
than
RR
T-P
CR
for
dete
ctin
gvi
ruse
s
Poul
try
7420
05
Con
tinue
don
follo
win
gpa
ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 433
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
Sam
plin
gen
viro
nmen
tV
irus
and/
ortr
acer
Part
icle
size
(�m
)A
naly
tical
met
hod
Com
men
tsA
eros
olso
urce
Ref
eren
ceY
rof
publ
icat
ion
AG
I-30
Set
2ex
peri
men
ts,
12.5
liter
s/m
inC
lose
dsy
stem
Bac
teri
opha
ges
MS2
and
T3
Part
icle
size
was
influ
ence
dm
ainl
yby
the
prop
ertie
sof
the
liqui
dm
ediu
man
dth
em
etho
dof
aero
soliz
atio
n,no
tby
the
phys
ical
size
ofth
evi
ruse
s;th
epa
rtic
les
stud
ied
wer
eun
der
300
nmin
diam
eter
Set
4ex
peri
men
ts,
cultu
reT
heca
ptur
eef
ficie
ncy
for
part
icle
sin
the
30-
to10
0-nm
size
rang
ew
as10
%or
low
er;t
heef
ficie
ncy
incr
ease
dfo
rpa
rtic
les
smal
ler
than
30nm
and
larg
erth
an10
0nm
;al
lthr
eesa
mpl
ers
exhi
bite
dlo
wca
ptur
eef
ficie
ncie
sfo
rul
trafi
nepa
rtic
les
that
vari
edov
ertim
e,as
wel
las
apo
tent
iall
oss
ofvi
rus
viab
ility
duri
ngsa
mpl
ing
Con
stan
tou
tput
atom
izer
7620
05
SKC
Bio
Sam
pler
Set
2ex
peri
men
ts,
12.5
liter
s/m
inC
lose
dsy
stem
Bac
teri
opha
ges
MS2
and
T3
Part
icle
size
was
influ
ence
dm
ainl
yby
the
prop
ertie
sof
the
liqui
dm
ediu
man
dth
em
etho
dof
aero
soliz
atio
n,no
tby
the
phys
ical
size
ofth
evi
ruse
s;th
epa
rtic
les
stud
ied
wer
eun
der
300
nmin
diam
eter
Set
4ex
peri
men
ts,
cultu
reT
heca
ptur
eef
ficie
ncy
for
part
icle
sin
the
30-
to10
0-nm
size
rang
ew
as10
%or
low
er;t
heef
ficie
ncy
incr
ease
dfo
rpa
rtic
les
smal
ler
than
30nm
and
larg
erth
an10
0nm
;al
lthr
eesa
mpl
ers
exhi
bite
dlo
wca
ptur
eef
ficie
ncie
sfo
rul
trafi
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rtic
les
that
vari
edov
ertim
e,as
wel
las
apo
tent
iall
oss
ofvi
rus
viab
ility
duri
ngsa
mpl
ing
Con
stan
tou
tput
atom
izer
7620
05
Fri
tbu
bble
rSe
t2
expe
rim
ents
,12
.5lit
ers/
min
Clo
sed
syst
emB
acte
riop
hage
sM
S2an
dT
3Pa
rtic
lesi
zew
asin
fluen
ced
mai
nly
byth
epr
oper
ties
ofth
eliq
uid
med
ium
and
the
met
hod
ofae
roso
lizat
ion,
not
byth
eph
ysic
alsi
zeof
the
viru
ses;
the
part
icle
sst
udie
dw
ere
unde
r30
0nm
indi
amet
er
Set
4ex
peri
men
ts,
cultu
reT
heca
ptur
eef
ficie
ncy
for
part
icle
sin
the
30-
to10
0-nm
size
rang
ew
as10
%or
low
er;t
heef
ficie
ncy
incr
ease
dfo
rpa
rtic
les
smal
ler
than
30nm
and
larg
erth
an10
0nm
;al
lthr
eesa
mpl
ers
exhi
bite
dlo
wca
ptur
eef
ficie
ncie
sfo
rul
trafi
nepa
rtic
les
that
vari
edov
ertim
e,as
wel
las
apo
tent
iall
oss
ofvi
rus
viab
ility
duri
ngsa
mpl
ing
Con
stan
tou
tput
atom
izer
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VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 435
on May 8, 2020 by guest
http://mm
br.asm.org/
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nloaded from
TA
BL
E2—
Con
tinue
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Sam
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type
Air
sam
plin
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teor
capa
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plin
gen
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size
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AG
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Cul
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quan
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RT
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the
AG
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mpl
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d20
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mor
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30an
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quan
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RT
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dA
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mer
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ruse
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anth
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4sa
mpl
erat
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anth
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30an
dA
GI-
4sa
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at15
and
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ollis
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orne
viru
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pone
ntia
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436 VERREAULT ET AL. MICROBIOL. MOL. BIOL. REV.
on May 8, 2020 by guest
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rus
and
RN
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pend
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ofth
esa
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met
hod
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May
sam
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rsa
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each
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and
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six
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xim
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rus
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20(n
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ture
and
RR
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num
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fect
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rus
and
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ofth
esa
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num
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inde
pend
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ofth
esa
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ing
met
hod
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25-m
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ters
with
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hala
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ampl
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elat
infil
ter
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liter
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teri
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irbo
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icle
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entr
atio
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ere
mea
sure
ddo
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man
dup
stre
amfr
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ters
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Phys
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x-je
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jet
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ter
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ber
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icle
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due
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high
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drop
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jet
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lison
-typ
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r-je
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buliz
er23
2007
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ter
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coun
ters
(air
born
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rtic
leco
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trat
ions
wer
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nstr
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and
upst
ream
from
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filte
rs)
Phys
ical
colle
ctio
nef
ficie
ncy
was
68%
Six-
jet
Col
lison
-typ
eai
r-je
tne
buliz
er23
2007
Con
tinue
don
follo
win
gpa
ge
VOL. 72, 2008 SAMPLING OF AIRBORNE VIRUSES 437
on May 8, 2020 by guest
http://mm
br.asm.org/
Dow
nloaded from
TA
BL
E2—
Con
tinue
d
Sam
pler
type
Air
sam
plin
gra
teor
capa
city
Sam
plin
gen
viro
nmen
tV
irus
and/
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acer
Part
icle
size
(�m
)A
naly
tical
met
hod
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men
tsA
eros
olso
urce
Ref
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ceY
rof
publ
icat
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PCfil
ter
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min
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mbe
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riop
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icle
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ters
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born
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rtic
leco
ncen
trat
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wer
em
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nstr
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filte
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ical
colle
ctio
nef
ficie
ncy
was
27%
Six-
jet
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lison
-typ
eai
r-je
tne
buliz
er23
2007
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ter
(0.5
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4lit
ers/
min
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mbe
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acte
riop
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10–8
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icle
coun
ters
(air
born
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rtic
leco
ncen
trat
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wer
em
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nstr
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and
upst
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from
the
filte
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Mor
eth
an96
%ph
ysic
alco
llect
ion
effic
ienc
y
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lison
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r-je
tne
buliz
er23
2007
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Efil
ter
(1-�
mpo
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4lit
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min
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mbe
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acte
riop
hage
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10–8
0nm
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icle
coun
ters
(air
born
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rtic
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trat
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nstr
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from
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alco
llect
ion
effic
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y
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lison
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r-je
tne
buliz
er23
2007
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Efil
ter
(3-�
mpo
resi
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ers/
min
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mbe
rB
acte
riop
hage
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10–8
0nm
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icle
coun
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(air
born
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rtic
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ncen
trat
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and
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from
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eth
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alco
llect
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effic
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lison
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r-je
tne
buliz
er23
2007
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thre
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rt37
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ticca
sset
tes
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ter
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2lit
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min
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mbe
rB
acte
riop
hage
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10–8
0nm
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icle
coun
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(air
born
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rtic
leco
ncen
trat
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wer
em
easu
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dow
nstr
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and
upst
ream
from
the
filte
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Mor
eth
an96
%ph
ysic
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llect
ion
effic
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lison
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r-je
tne
buliz
er23
2007
SKC
Bio
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pler
impi
nger
s12
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ers/
min
for
1m
in13
3-lit
erst
ainl
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stee
ldyn
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aero
solt
oroi
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hoda
min
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roin
fect
ivity
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pera
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had
agr
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ref
fect
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onth
est
abili
tyof
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24-J
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onne
buliz
er72
2007
Bio
aero
solp
erso
nal
sam
pler
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for
5m
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olch
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fluen
zavi
rus
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rain
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ture
and
RR
T-
PCR
Vir
uses
wer
ede
tect
edin
aero
sols
ampl
esT
hree
-jet
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lison
nebu
lizer
111
2007
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mge
latin
filte
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ers/
min
for
5or
10m
in3.
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by4.
6-m
by3.
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ber
with
uppe
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obstacle. This is the only mechanism that does not depend onparticles being diverted from the streamline. Inertial impactionoccurs when a particle impacts an obstacle when the streamlinechanges direction. The inertia of the particle forces it to divertfrom the streamline and to impact a surface. As mentionedpreviously, only very small particles are affected by the diffu-sion mechanism based on Brownian motion. Gravitational set-tling affects mostly particles of much larger aerodynamic di-ameter by pushing them downward due to gravity. Theimportance of this force depends on the other forces affectingthe particle in various directions. Lastly, electrostatic forcesalso influence the trajectory of particles. This mechanism de-pends on the size and charge of the particle and the chargedifference with the filter.
Given that small particles are highly governed by the diffu-sion phenomenon and that larger particles have a tendency toimpact and to be intercepted, it was shown that both types ofbehaviors are at their lowest at 0.3 �m (75). Thus, filters areleast efficient at removing 0.3-�m particles. Filtration efficiencyimproves with increasing and decreasing particle size. This iswhy filter efficiency is based on the 0.3-�m benchmark.
Many different types of filters have been used to sampleairborne viruses. They differ mainly in composition, pore size,and thickness. To our knowledge, the first filters used to sam-ple airborne viruses were made out of tightly packed cottonand were used to sample variola virus (Poxviridae family;dsDNA virus) in a hospital (95). Cellulose filters (0.45-�mpore size) have also been used to sample hospital air; PCRanalysis of these samples permitted the detection of naturallyproduced aerosols of varicella-zoster virus (Herpesviridae fam-ily; dsDNA virus; 200 nm) (117) and respiratory syncytial virus(Paramyxoviridae family; ssRNA virus; 150 nm) (3). PTFE fil-ters (2.0-�m pore size) have been used to collect artificialrhinovirus (Picornaviridae family; ssRNA virus; 25 to 30 nm)aerosols in a small aerosol chamber (101) and naturally pro-duced rhinovirus aerosols in office buildings (102). In bothcases, PCR was used to detect the viruses. While polycarbon-ate filters are much less efficient than gelatin or PTFE filters(23), 0.1-�m polycarbonate filters have been used in combina-tion with PCR to detect human cytomegalovirus (Herpesviridaefamily; dsDNA virus; 200 nm) in samples of naturally producedaerosols (92). The low filtration efficiency of polycarbonatefilters may be due to the structure of the filter. The contactarea of filters with uniform cylindrical pores, such as polycar-bonate filters, is much smaller than that of filters with a com-plex structure, such as PTFE filters, where the probability ofadherence is greater because airborne particles are exposed toa greater surface area.
However, filters are not commonly used to sample airborneviruses because they can cause structural damage. In addition,the desiccation of the samples that occurs during sampling caninterfere with culture analysis of the samples. While modernmolecular biology tools do not require infectious particles todetect viruses, studies investigating the effects of environmen-tal factors on viral infectivity, for example, require the collec-tion of infectious viruses. Gelatin filters can be used becausethey do not appear to significantly affect viral infectivity. Forexample, MD8 air samplers equipped with 80-mm gelatinmembrane filters with a pore size of 3 �m in combination withculture techniques have been used successfully to detect Lac-
tococcus lactis tailed bacteriophages in a cheese factory (103).Gelatin filters, as well as the Andersen sampler and theAGI-30 impinger, are 10 times more efficient than polycarbon-ate filters at collecting active bacteriophages (132). The phys-ical collection efficiencies of both gelatin and PTFE filters,calculated by placing particle counters before and after thefilters, exceed 96% (23). While gelatin filters can be very usefulfor sampling functional viruses, their use can be limited byenvironmental conditions. Low humidity can cause them to dryout and break, while high humidity or water droplets can causethem to dissolve. On the positive side, this property can beused to recover viruses or virus-laden particles by dissolvingthe filters in water. Nevertheless, 0.3-�m PTFE filters appearto be the best option for long-term sampling of 10- to 900-nm-diameter virus-laden particles (23).
Filter materials mounted on three-piece cassettes all havethe same limitation. These cassettes are hollow cylinders madeout of plastic or metal, with an inlet or outlet hole at the centerof the base of each cylinder. A filter is deposited on a poroussupport pad (cellulose, plastic, or metal) on part one, the base.The second part, the cylinder, is placed on the base to seal theedge of the filter and ensure that the air pumped from theoutlet of the cassette passes through the filter. If only the firsttwo parts are used, it is considered an open cassette. The thirdpart is placed on top to close the cassette. The pressure used toseal the cassettes can influence the outcome of an experiment.If the cassette is not properly sealed, aerosol slippage canoccur. The airflow goes around the filter and into the pump,preventing most of the airborne particles from being capturedby the filter and significantly contaminating the pump in theprocess. On the other hand, if the cassette is sealed too tightly,the filter can be damaged or weakened, especially with fragilefilters such as gelatin membranes, and aerosol slippage can alsooccur (135). If premounted cassettes are available with thedesired filters, their efficiency should be compared to that oflaboratory-prepared cassettes.
Standardized filters are still available but are often left asidebecause of the damage they can cause to viruses. In compari-son studies, filters often provide the best physical recovery ofnanoscale particles, but the filtering process can damage vi-ruses and complicate the analysis, especially if culture is usedto assess virus levels. Comparative studies using culture as ananalytical tool have shown that filters are less efficient thanother, less destructive methods, such as liquid impingers, forrecovering airborne infectious viruses (64, 66, 132).
Electrostatic Precipitators
LVS use electrostatic precipitation to sample air. One ex-ample is the LVS designed for the U.S. army in the 1960s (54).This device can draw up to 10,000 liters of air per minutethrough a high-voltage corona, where the particles are chargedbefore being precipitated onto a grounded rotating disc. Arecirculating fluid is used to wash off and concentrate theprecipitated particles. LVS have been used to recover airborneadenoviruses (Adenoviridae family; nonenveloped dsDNA vi-rus; 70 to 90 nm in diameter) in a military hospital, where50,545 liters of air were sampled in 5 minutes, with the partic-ulate content collected in 180 ml of fluid (11). LVS have alsobeen used in other circumstances to recover low concentra-
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tions of airborne adenoviruses and picornaviruses (12, 13, 38,40, 51, 77, 94, 97, 121, 144). These samplers are commonlyused with a preimpactor attached to the air inlet to captureparticles of over 15 �m in diameter. Smaller particles aresubsequently collected by electrostatic precipitation (12). Al-though LVS recover slightly more infective viruses than doesthe cyclone separator described by Errington and Powell (49),LVS are more complicated to operate (38). In addition, theproduction of ozone at high RH in the presence of the intenseelectric field may damage viruses (28).
Other Sampling and Detection Methods
Other methods can be used to detect airborne viruses with-out any actual air sampling. Swabbing the surfaces of air pu-rifier filters (44, 123) can be used to assess the viral composi-tion of air. Settling dishes (14, 40, 41, 126) can also be used, butthis method is more suitable for larger droplets that settle bygravity on a petri dish. While cumbersome, sentinel animalscan be used to detect the presence of viruses (21, 57, 105, 129).Susceptible host animals can be used as air samplers and cul-ture support systems (90) by exposing the animals to poten-tially contaminated air and then noting symptoms or perform-ing laboratory tests. Sufficient concentrations of airborne virusand appropriate conditions are required for this approach todetect viruses.
ASSESSING THE EFFICIENCY OF AIRBORNE VIRUSSAMPLERS BY USE OF TRACERS
In order to study the efficiency of samplers for airborne virussampling, a variety of strategies have been used. It is importantto note the difference between the capture efficiency of a sam-pler and its efficiency for viral recovery. The capture efficiency(or total physical efficiency) is based on the rate of recovery ofdifferent particle sizes and is measured with methods indepen-dent of the integrity of the viruses, while the efficiency of viralrecovery is, in most studies, an indicator of the remainingintegrity and infectivity of the sampled viruses.
Tracers can be used to measure the total capture efficiencyof samplers. P-32 (16, 65, 66), uranine (fluorescein sodium salt)(2, 32, 54, 55, 78, 81, 82, 84, 101), rhodamine B (46–48, 72, 116,122), and Bacillus spores (17, 77, 119) have all been used astracers to detect airborne viruses. Uranine remains the mostpopular tracer because it is safer than radiolabeling for aerosolstudies. In addition, unlike rhodamine B, it does not affect viralinfectivity (79). New molecular methods have led to alternativetracers for determining sampler efficiency. For example, viralgenetic material can be used to estimate the total number ofviruses sampled. Genomic tracers do not depend on viral in-fectivity or, to a certain degree, viral integrity. However, thelack of information on the degradation of viral genetic materialin aerosols makes the replacement of physical tracers by quan-titative PCR premature (72). The major weakness of the quan-titative PCR method is the detection limit. Lastly, particlecounters can be installed upstream and downstream from thesampler to determine the total number of particles trapped bythe sampler. However, this method cannot be used to deter-mine the proportion of captured particles that can be extractedfor further analysis.
LABORATORY STUDIES OF AIRBORNE VIRUSES
To our knowledge, the oldest study on the sampling of air-borne viruses was performed with a laboratory setup using achamber and an artificially produced aerosol of influenza virus(140). Since then, many chamber setups have been used tostudy artificially produced infectious aerosols (61, 70, 139) andairborne viruses. Rotating drum or dynamic aerosol toroids(61) have been used to study the biological decay rates ofairborne viruses under different temperature and/or RH con-ditions (4, 5, 16, 42, 46–48, 65, 67, 72, 78, 80–82, 84, 116, 119,122, 138). These devices make it possible to study aerosolsunder controlled atmospheric conditions for extended periods,with little loss of airborne particles to gravitational settling. Avariety of chambers and other types of closed and/or controlledsystems, some inspired by previous technologies, have beenused to study artificially and naturally produced aerosols (2, 7,8, 15, 20, 23, 30–33, 38, 39, 45, 54–58, 60, 62, 64, 66, 73, 76, 86,99, 101, 107, 109, 118, 120, 121, 130–133, 137). Most setups forviral aerosol studies are handmade for specific purposes. Theaerosol source or generator, temperature, RH, radiation, timeof exposure, aerosolization medium, sampling method, viruses,tracers, and analytical methods are rarely the same. As such,even controlled studies are difficult to compare.
Aerosol generators are most often used to study the behav-ior of airborne viruses. For the generation of submicrometeraerosols, neutralizers are placed between the generator andthe chamber to prevent uncommon aerosol behavior by remov-ing charges on particles created during the nebulization pro-cess. Desiccators are also often used to shrink particles byevaporation. The median size of aerosol particles is controlledby the intensity of the aerosolization process and by preimpac-tors to stop larger particles. Since the concentration of thenonevaporative solutes in the nebulization medium determinesthe size of the droplet nuclei, low solute concentrations can beused to produce small particles and high solute concentrationscan be used for large particles. The concentration of the aero-sol in the chamber can be modified by adding clean dilution air.The size of the droplet nuclei can be calculated using equationsthat take into account the initial droplet diameter and thevolume fraction of solid material (75).
Many types of devices can be used to determine the concen-tration of particles in the air. Spectrometers equipped withvarious technologies can count and size airborne particles inreal time or near real time. However, they are often limited tomeasuring particles of �300 nm to 500 nm in diameter. Scan-ning mobility particle sizers can be used to count and measuresmaller particles. These devices neutralize airborne particlesbefore separating them based on electrical mobility (chargeand size). The particles are then passed through a condenser toincrease their size so they can be detected by photometry.
SURROGATE VIRUSES
While aerobiological studies using hazardous viruses canprovide valuable information, they can also expose personnelto unnecessary risks. Surrogate viruses, such as bacteriophagesof E. coli and other bacteria, can be used to mimic the behaviorof pathogenic viruses. Bacteriophages of the order Caudovi-rales, such as T7-like (16, 30, 42, 45, 67, 76, 99, 122, 131–133,
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137, 138), T1-like (64, 66), and T5-like (69) bacteriophages,were the first surrogates used. The genomic material of thesebacteriophages consists of dsDNA, the capsid is nonenveloped,and the presence of a tail for host recognition renders theviruses susceptible to physical damage. They are thus unreli-able for infectivity assays. In addition, the morphological char-acteristics of these bacteriophages do not resemble those ofany mammalian viruses. As such, they are used more rarelynowadays and have largely been replaced by other surrogateviruses. Bacteriophage MS2 (Leviviridae family) has been usedas a surrogate virus in many studies (14, 23, 42, 62, 76, 130, 132,133). It is a nonenveloped ssRNA coliphage with a very small(25 to 27 nm) icosahedral capsid. It has no tail and is morpho-logically similar to members of the Picornaviridae family, whichincludes many pathogenic viruses, such as poliovirus, rhinovi-rus, and FMD virus. Another surrogate virus is bacteriophage�X174 (Microviridae family), which possesses a ssDNA ge-nome and a morphology similar to that of MS2. Bacteriophage�X174 has been compared to the most resistant human-patho-genic viruses, such as polioviruses and parvoviruses (114), andhas been used in aerobiology (96, 132, 133) along with anothermicrovirus, S13 (42, 137). Bacteriophage �6 (Cystoviridae fam-ily; dsRNA virus; 85 nm) can be used as a surrogate for smallenveloped viruses (132, 133).
Since every virus has a unique response to environmentalfactors, no surrogate is perfect. Nonetheless, nonpathogenicmodels can greatly simplify virus studies, especially when aero-sols are used.
CONCLUSIONS
Sampling techniques have been improved greatly over theyears, and we are definitely better equipped today to tackle theimportant health issue of airborne viruses. However, the lackof standardization has to be addressed, as it limits the devel-opment of general recommendations for sampling of airborneviruses. Given the wide range of aerodynamic properties ofairborne viruses, which can be nanometer- to micrometer-sizedparticles, the issue of standardization is of the utmost impor-tance. The detection of viruses in air samples depends on thetype of aerosol and the sampling and analytical methodologies.Studies to date have rarely included quantitative analyses oftotal viral load. While culture is often used to determine virusconcentrations, most sampling methods affect viral infectivity,making culture inadequate for calculating the true concentra-tions of infectious airborne viruses. Technologies such as PCRcan be used to detect viruses in air samples even when they areno longer infectious. While filters cause more damage to vi-ruses than other methods do, they are more efficient for de-termining viral loads in aerosols. Lastly, viruses are an aston-ishingly diverse group of microorganisms, so this diversity hasto be taken into consideration to select the most appropriatesampling devices. Whatever techniques or recommendationsare proposed for sampling virus aerosols, it is important tokeep in mind that a representative sample should containnanoparticles together with larger airborne particles. Futurestudies will contribute to better predicting the potential risk ofinfection by airborne viruses.
ACKNOWLEDGMENTS
This study was funded by a concerted grant from FQRNT-NOVALAIT-MAPAQ and by Agriculture and Agri-Food Canada.S.M. and C.D. also received funding from the Natural Sciences andEngineering Research Council of Canada (NSERC). C.D. is therecipient of an FRSQ Junior 2 scholarship.
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