Airborne Virus Capture and Inactivation by an Electrostatic Particle Collector E R I C M . K E T T L E S O N , ��� B A L A R A M A S W A M I , ��� C H R I S T O P H E R J . H O G A N , J R . , ��� M Y O N G - H W A L E E , ��� G E N N A D I Y A . S T A T Y U K H A , �� P R A T I M B I S W A S , ��� A N D L A R G U S T . A N G E N E N T * , | Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, Environmental and Energy Division, Korea Institute of Industrial Technology, CheonAn City, South Korea, Cybernetics of Chemical Technology Processes, National Technical University of Ukraine, Kiev, Ukraine, and Department of Biological and Environmental Engineering, Cornell University, 214 Riley-Robb Hall, Ithaca, New York 14853 Received November 20, 2008. Revised manuscript received May 19, 2009. Accepted June 2, 2009. Airborne virus capture and inactivation were studied in an electrostatic precipitator (ESP) at applied voltages from -10 to +10 kV using aerosolized bacteriophages T3 and MS2. For each charging scenario, samples were collected from the effluent air stream and assayed for viable phages using plaque assays and for nucleic acids using quantitative polymerase chain reaction (qPCR) assays. At higher applied voltages, more virus particles were captured from air with maximum log reductions of 6.8 and 6.3 for the plaque assay and 4.2 and 3.5 for the qPCR assay at -10 kV for T3 and MS2, respectively. Beyond corona inception (i.e., at applied voltages of -10, -8, +8, and +10 kV), log reduction values obtained with the plaque assay were much higher compared to those of the qPCR assay because nonviable particles, while present in the effluent,wereunaccountedforintheplaqueassay.Comparisons oftheseassaysshowedthatin-flightinactivation(i.e.,inactivation without capture) was greater for the highest applied voltages with a log inactivation of 2.6 for both phages at -10 kV. We have demonstrated great potential for virus capture and inactivation via continual ion and reactive species bombardment when conditions in the ESP are enforced to generate a corona discharge. Introduction According to the World Health Organization, the most prevalent transmissible diseases in the world are respiratory infections (1). These infections, originating from bacterial or viral exposure, may be transmitted in indoor environments via a number of different ways, including direct or indirect contact with an infected surface, droplet transmission, or aerosol transmission (2). Aerosol transmission is defined as host inhalation of infectious nuclei ( 5 ��m in diameter) that have remained suspended in air and disseminated through- out an indoor environment (3). Considering that the average person in the United States spends nearly 87% of his time indoors (4), maintaining a clean indoor-air environment is an important avenue for mitigating aerosol transmission of respiratory infections. Engineering controls employed in air handling systems aim to effectively collect/remove aerosol particles from the air stream, utilizing various capture mechanisms depending onthetechnologyused.High-efficiencyparticulateair(HEPA) filtration can be used to remove airborne particles of biological origin (i.e., bioaerosols) in many indoor environ- ments, including hospitals, office buildings, and aircraft cabins. However, implementation of HEPA filtration bears excessive operational costs because of regular filter replace- mentandadditionalpowerrequirementsforairrecirculation due to a large pressure drop across the filter material. In addition, the filter itself may provide a growth habitat for bacteria (5). Coal-fired power plants have circumvented the excessive operational costs by employing electrostatic pre- cipitators (ESPs) to efficiently control fine, nonbiological particle emissions. ESPs have also been placed in household air-forced heating and cooling systems to control aerosol levels. Recently, this particle collection technology has been extensivelypromotedasastand-alone���airpurifier���tocontrol household bioaerosols. ESPs consist of charging and collecting electrodes with a negative or positive potential difference between them to create an electric field. Typically, a high voltage is applied to a thin wire (acting as the central charging electrode) to generate ions in an electric field, which collide with and charge the aerosol particles (6). Charged aerosol particles migrate toward the oppositely charged collecting electrode, which typically consists of a stainless-steel cylinder or plate. The magnitude of the attractive force on charged particles isproportionaltothevoltageappliedtothechargingelectrode (7).Atlowtomoderateappliedvoltages,onlysmallquantities of electrons flow to the positive electrode. As the applied voltage increases, the corresponding increase in electric field strength accelerates the flow of electrons. When sufficient kinetic energy is gathered, the collision of an electron with an atom or a gas molecule produces an ion and an additional free electron. Further collisions will generate more ions and free electrons, eventually leading to an exponential increase in the number of free electrons. The high level of ions associatedwiththisphenomenonischaracteristicofacorona discharge (8), which allows for efficient charging and capture of airborne particles. The usefulness of ESP technology in mitigating biological aerosols has been demonstrated using both bacterial en- dospores and various bacterial species (9, 10). Other pub- lished research involving electrostatic precipitation of bio- aerosols has focused almost exclusively on the sampling efficiency of bacterial cells and spores for exposure assess- ment and their survival rates (i.e., bioavailability) after electricalcharging(11-15).Similarly,thefewstudiesonvirus particle behavior in ESPs have only focused on sampling efficiency and not on mitigation effectiveness (16-18). In addition, a recent paper has combined ESP with biosensors to monitor airborne virus particles (19). ESPs have size- dependent collection efficiencies, and while overall mass- based collection efficiencies may be high (e.g., 99%), the collection efficiencies of particles in the submicrometer and * Corresponding author tel: +1-607-255-2480 fax: +1-607-255- 4080 e-mail: la249@cornell.edu. ��� Washington University in St. Louis. ��� Korea Institute of Industrial Technology. �� National Technical University of Ukraine. | Cornell University. Environ. Sci. Technol. 2009, 43, 5940���5946 5940 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 15, 2009 10.1021/es803289w CCC: $40.75 ��� 2009 American Chemical Society Published on Web 06/23/2009

nanometerparticlesizerangearetypicallylow(20-23).Many virus particles are in the submicrometer and nanometer size ranges. The potential to capture such biological particles with high efficiency, and potentially inactivate them in an ESP system has considerable practical importance. Besides our initial report on physical measurements of particle capture (24), the effectiveness of ESPs as an engineering mitigation control for inactivation of airborne virus particles has not been investigated. Thus, the objective of this study was to quantify the capture and inactivation of airbornevirusparticlesusingelectrostaticprecipitationunder different operating conditions. To further understand virus collection and inactivation, a soft X-ray enhanced ESP was used in this study (20, 24, 25). Previous research has shown that nonbiological nanoparticles (i.e., particles up to 100 nm in size) are difficult to charge in conventional ESPs (23), and that soft X-ray irradiation aids in charging such particles by producing additional bipolar ions and also by direct pho- toionization (20, 26). Respiratory infection in humans can be caused by a diverse assortment of viruses, and therefore we propagated and aerosolized both dsDNA and ssRNA viruses, bacteriophages T3 and MS2, with diameters of 45 and 25 nm, respectively. Two assays were used to quantify reduction of bacteriophages T3 and MS2 from the aerosol stream:aplaqueassay,whichmeasuredthenumberofviable (i.e., plaque forming) phages in the effluent aerosol and a qPCR assay, which quantified the total nucleic acid mass for both active and inactive T3 and MS2 in the effluent aerosol. PCR-basedmethodsforquantifyingairbornemicroorganisms have been used to assay aerosol samples (27), and have been incorporated in recent work relating to bacterial (28) and viral aerosols (29). From the plaque and qPCR assays, we determined the number of viable phages relative to total phages exiting the system, thereby distinguishing between physical capture and inactivation. Based on these results, mechanismsofviralinactivationwithintheESParediscussed. Materials and Methods PropagationofBacteriophagesT3andMS2. Bacteriophages T3 (ATCC 11303-B3) and MS2 (ATCC 15597-B1) were propagated using Escherichia coli strain C (ATCC 13706) and strain C-3000 (ATCC 15597) as their respective hosts. The growth media used for E. coli strain C was LB broth (Sigma- Aldrich, St. Louis, MO) amended with CaCl2 (Fisher, Pitts- burgh, PA) and MgSO4 (Sigma-Aldrich) [per L of deionized water: 20 g of LB broth, 10 mL of 1.0 M MgSO4, and 1.5 mL of 1.0 M CaCl2], while minimal media [per L of deionized water: 100 mL of 10X M9 mix, 10 mL of 10% glucose (Fisher) 10 mL of 1% thiamine (Sigma-Aldrich), and 1 mL of 1.0 M MgSO4] was used to culture Escherichia coli strain C-3000. To generate the necessary large-volume, high-titer bacte- riophage stock solutions, a 3-day liquid-culture technique (see Supporting Information (SI)) was used. We prepared single high-titer stock solutions for both bacteriophages T3 andMS2tomaintainconsistencybetweenexperiments.Stock solution concentrations were 1.2 �� 109 PFU mL-1 and 2.0 �� 1010 PFU mL-1 for bacteriophages T3 and MS2, respectively. The stock solutions were prepared at the beginning of the study and stored at 4 ��C. Before every experimental run, 10 mL of the stock solution was mixed with 55 mL of DI water to make a diluted working solution (1.8 �� 108 PFU mL-1 and 3.1 �� 109 PFU mL-1 for bacteriophages T3 and MS2, respectively), which would then be used for aerosolization. Aerosolization. Different aerosolization techniques were used for T3 and MS2 bacteriophages: a constant output stainless steel atomizer was used for bacteriophage T3 to preventexcessivefoamingofitsspecificbroth a6-jetCollison nebulizer was used for bacteriophage MS2 to maintain consistency between this study and our preliminary work with MS2 (24). Bacteriophage T3 was aerosolized using a constant output atomizer (model 3076, TSI, Inc., St. Paul, MN) containing 65 mL of bacteriophage T3 working solution (Figure 1, path A). The atomizer was operated at an upstream pressureof240kPaataflowrateof3.0Lmin-1,andgenerated an aerosol with an average number concentration of 9.3 �� 106 particles cm-3, a geometric mean diameter (GMD) of 41.3 nm, and a geometric standard deviation (GSD) of 1.82. Particle number concentration, GMD, and GSD were mea- sured using a differential mobility analyzer (model 3081 in Electrostatic Classifier model 3080, TSI Inc., Minneapolis, MN) and an ultrafine condensation particle counter (model 3022a, TSI Inc.) operating together as a scanning mobility particle spectrometer (30). HEPA-filtered compressed air at a flow rate of 9.5 L min-1 was bubbled through a glass impinger (Ace Glass Inc., Vineland, NJ) containing 10 mL of DI water and combined with the output of the atomizer to further humidify and dilute the air stream to prevent drying and inactivating bacteriophage T3 (31). The bacteriophage- laden, humidified aerosol stream was then delivered at a total flow rate of 12.5 L min-1 to the inlet of a soft X-ray FIGURE 1. Experimental setup for the aerosolization and electrostatic treatment of bacteriophages T3 (path A) and MS2 (path B). Total particle concentration measurements of the ESP effluent air stream are depicted by dashed lines. VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5941