Improving Protection against Viral Aerosols through Development of Novel Decontamination Methods and Characterization of...

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Title:
Improving Protection against Viral Aerosols through Development of Novel Decontamination Methods and Characterization of Viral Aerosols
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1 online resource (163 p.)
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english
Creator:
Woo, Myung Heui
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Environmental Engineering Sciences
Committee Chair:
Wu, Chang-Yu
Committee Members:
Koopman, Ben L
Sigmund, Wolfgang M
Wander, Joseph D

Subjects

Subjects / Keywords:
bioaerosol -- charaterization -- decontamination -- inactivation -- ms2 -- survivability
Environmental Engineering Sciences -- Dissertations, Academic -- UF
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Environmental Engineering Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Although respirators and filters are designed to prevent the spread of pathogenic aerosols, a stockpile shortage is anticipated during the next flu pandemic. Contact transfer and reaerosolization of collected microbes from used respirators are also a concern. An option to address these potential problems is to decontaminate used respirators/filters for reuse. In this research, a droplet/aerosol loading chamber was built and applied for decontamination testing for fair comparison of the performance of different decontamination techniques. Different decontamination techniques including antimicrobial chemical agents, microwave irradiation, ultraviolet irradiation, were incorporated into the filtration system and evaluated. As biocidal filter, dialdehyde cellulose/starch filters were investigated for their inactivation efficacy. Both filters with sufficient moisture content had a higher removal efficiency, lower pressure drop, and better disinfection capability, which are all important attributes for practical biocidal applications. For microwave-assisted filtration system, temperature was identified to be a key factor. Relative humidity was another pivotal parameter for viability of viruses at warm-to-hot-water temperatures, but it became insignificant at temperatures above 90 degrees C. As environmental conditions greatly affect the viability of microorganism, the effect of relative humidity and spray medium on UV inactivation was examined. Absorption of UV by high water content and shielding of viruses near the center of the aggregate are considered responsible for lower inactivation. Across different spray media, inactivation efficiencies in artificial saliva (AS) and in beef extract (BE) were much lower than in deionized water for both aerosol and droplet transmission, indicating that solids present in AS and BE exhibited a protective effect. For particles sprayed in a protective medium, relative humidity was not a significant parameter. The distribution of infectious MS2 aerosols followed volume-based size distribution for pure viral aerosol whereas these of infectious MS2 generated with solid contents followed lower dimensions. Aggregation by MS2 itself and encasement by inert salts yielded higher stability factor because of shielding effect and reduction of the air/water interface whereas the soluble salts resulted in adverse effect. The knowledge and technologies developed in this study can better protect the general public as well as healthcare facilities against viral aerosols.
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In the series University of Florida Digital Collections.
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Includes vita.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Myung Heui Woo.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Wu, Chang-Yu.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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1 IMPROV ING PROTECTION AGAINST VIRAL AEROSOLS THROUGH DEVELOPMENT OF NOVEL DECONTAMINATION METHODS AND CHARACTERIZATION OF VIRAL AEROSOL By MYUNG HEUI WOO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 201 2 Myung Heui Woo

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3 To my family in Korea for their constant love and support

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4 ACKNOWLEDGMENTS I could not have accompl ished this study without the valuable assistance of many individuals. Here, I would like to express my deep appreciation to those people. I gratefully acknowledge my advisor, Dr. Chang Yu Wu for the introduction of aerosol fi e l d and giving me an opportunit y to join his group for bioaerosol study. His guidance, knowledge, encourage ment critical thinking, and even writing skill have inspired me for research work. I especially appreciate Dr. Josep h Wander who provide s valuable comments based on the wide and i n depth knowledge of bioaerosol to improve this study. I wish to express my gratitude to Drs. Ronald Baney and Wolfgang Sigmund for providing the filter mat erials and valuable comments on filter characteristics. I am also grateful to my committee member, D r. Ben Koopmen for reading this dissertation and providing the priceless comments. Great appreciation should go to Dr. Myoseon Jang for guidance and advice for the present and future research fields I also thank Brian Heimbuch and Dr. William Wallace in Air Force Research Laboratory in Tyndall AFB for their guidance and advices. I also wish to acknowledge the staffs in MAIC PERC and ICBR who instruct me the use of instruments and helped me with operation I would like to thank all aerogators Dr. Jin Hwa Lee, Dr. Yu Mei Hsu, Lindsey Riemenschenider, Alex D. Theodore, Qi Zhang, Danielle Hall, Brian Damit, Seungo Kim, Dr. Sewon Oh, Jun Wang, Lin Shou, Nima A. Mohajer, Dr. Hsing Wang Li, and Matt Tribby for their discussions and friendship Many thanks g o out to my undergraduate students, Kyle Brown, Kyle Ulmer, Diandra Anwar, Arian Tuchman, Sang Gyou Rho, Christiana Lee, Tammy Smith, and Adam Grippin for their help s for my research My special thanks go to my friends Drs. Youngmin Cho, Sejin Youn, and H wan Chul Cho.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 LIST OF A CRONYMS ................................ ................................ ................................ ................. 12 ABSTRACT ................................ ................................ ................................ ................................ ... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 17 Biological Threat ................................ ................................ ................................ .................... 17 Viral A erosol ................................ ................................ ................................ .......................... 17 Transmission M ode ................................ ................................ ................................ ................ 18 Filtration ................................ ................................ ................................ ................................ 19 Decontamination M ethods ................................ ................................ ................................ ...... 20 Research Objectives ................................ ................................ ................................ ................ 22 2 METHOD FOR CONTAMINATION OF FILTERING FACEPIECE RESPIRATORS BY DEPOSITION OF MS2 VIRAL AEROSOLS ................................ ................................ 24 Background ................................ ................................ ................................ ............................. 24 Materials and Methods ................................ ................................ ................................ ........... 27 Virus and Nebulization Fluid Preparation ................................ ................................ ....... 27 Test Material ................................ ................................ ................................ .................... 27 Droplet/Aerosol Loading System ................................ ................................ .................... 28 Chamber O peration and D etermination of O perating C onditions ................................ ... 29 Statistical A nalysis ................................ ................................ ................................ .......... 31 Results and Discussion ................................ ................................ ................................ ........... 31 D etermination of O perati ng C onditions ................................ ................................ .......... 31 Droplet Size Distribution ................................ ................................ ................................ 33 Loading onto NIOSH certified FFRs ................................ ................................ .............. 35 Summary ................................ ................................ ................................ ................................ 36 3 EVALUATION OF THE PERFORMANCE OF DIALDEHYDE CELLULOSE FILTERS AGAINST AIRBORNE AND WATERBORNE BACTERIA AND VIRUSES ................................ ................................ ................................ ................................ 44 Backgroun d ................................ ................................ ................................ ............................. 44 Materials and Methods ................................ ................................ ................................ ........... 47 Test Filters ................................ ................................ ................................ ....................... 47

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6 Test Bacteria and Bacteriophages ................................ ................................ ................... 47 Water F iltration ................................ ................................ ................................ ............... 48 Air F iltration ................................ ................................ ................................ .................... 49 Removal Efficiency, Relative Survival Fraction and Statistical Analysis ...................... 50 Results ................................ ................................ ................................ ................................ ..... 51 Water F iltration ................................ ................................ ................................ ............... 51 Air F iltration ................................ ................................ ................................ .................... 52 Discussion ................................ ................................ ................................ ............................... 53 Water F iltration ................................ ................................ ................................ ............... 53 Air F iltration ................................ ................................ ................................ .................... 54 Summary ................................ ................................ ................................ ................................ 56 4 USE OF DIALDEHYDE STARCH TREATED FILTERS FOR PROTECTION AGAISNT AIRBORNE VI RUSES ................................ ................................ ........................ 67 Background ................................ ................................ ................................ ............................. 67 Materials and Methods ................................ ................................ ................................ ........... 68 Test Agent ................................ ................................ ................................ ....................... 69 Experimental Method ................................ ................................ ................................ ...... 69 Results and Discussion ................................ ................................ ................................ ........... 71 Summary ................................ ................................ ................................ ................................ 73 5 MICROWAVE IRRADIATION ASSISTED hvac FILTRATION FOR INACTIVATION OF VIRAL AEROSOLS ................................ ................................ .......... 80 Background ................................ ................................ ................................ ............................. 80 Materials and Methods ................................ ................................ ................................ ........... 82 Test Filters and Agent ................................ ................................ ................................ ..... 82 Experimental System ................................ ................................ ................................ ....... 82 Results and Discussion ................................ ................................ ................................ ........... 85 Temperature M easurement of T est F ilters ................................ ................................ ...... 85 Inactivation E ffic iency and S urvival F raction ................................ ................................ 86 Effective T emperature ................................ ................................ ................................ ..... 87 Effect of R elative H umidities on I nactivation P erformance ................................ ........... 89 Degradation of F ilters after M icrowave I rradiation ................................ ........................ 90 Comparison to Other Disinfection Technologies ................................ ............................ 91 Summary ................................ ................................ ................................ ................................ 92 6 E FFECTS OF RELATIVE HUMIDITIES AND AEROSOLIZED MEDIA ON uv DECONTAMINATION OF VIRAL AEROSOLS LOADED FILTER .............................. 102 Background ................................ ................................ ................................ ........................... 102 Materials and Methods ................................ ................................ ................................ ......... 105 MS2 Preparation ................................ ................................ ................................ ............ 105 Spraying M edium ................................ ................................ ................................ .......... 106 Droplet and A erosol L oading S ystem ................................ ................................ ........... 107 UV E xposure ................................ ................................ ................................ ................. 107

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7 Results and Discussion ................................ ................................ ................................ ......... 108 Effect of Transmission Mode with Different Media ................................ ..................... 108 Effect of RH du ring Bot h L oading and In ctivation ................................ ....................... 111 Virus Susceptibility ................................ ................................ ................................ ....... 113 Summary ................................ ................................ ................................ ............................... 114 7 E FFECTS OF RELATIVE HUMIDITIES AND S PRAY MEDIA ON S URVIABILITY OF VIRAL AEROSOLS ................................ ................................ ................................ ...... 123 Background ................................ ................................ ................................ ........................... 123 Materials and M ethods ................................ ................................ ................................ ......... 124 Test V irus and Spraying Medium ................................ ................................ .................. 124 Experimental D esign and T asks ................................ ................................ .................... 125 Results and discuss i on ................................ ................................ ................................ .......... 129 Collection E fficiency of BioSamplers ................................ ................................ ........... 129 Size D istribution of MS2 in D ifferent E nviro nmental C onditions ................................ 130 Size Distribution of I nfectious MS2 ................................ ................................ .............. 131 Summary ................................ ................................ ................................ ............................... 133 8 CONCLUSIONS AND RECOMMENDATIONS ................................ ............................... 141 APPENDIX A PRELIMINARY TEST FOR DROPLET LOADING CHAMBER ................................ ..... 144 B MICROWAVE ASSISTE D PAN_NANO FILTRATION SYSTEM FOR VIRAL AEROSOL ................................ ................................ ................................ ............................ 144 L IST OF REFERENCES ................................ ................................ ................................ ............. 155 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 163

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8 LIST OF TABLES Table page 2 1 Composition of artificial saliva (Based on 979 mL of DI water) ................................ ...... 37 2 2 Loading density and C Vs of Q T Q and S T S for 6 different FFRs (N = 3, Criteria of CV for Q T Q and S T S: 20% and 40% respectively ) ................................ ................. 37 3 1 Viable removal efficiency and relative sur vival fraction of untreated filter and three DAC filters treated under different treatment times in water filtration. ............................ 57 3 2 Pressure drop and quality factor based on physical removal effici ency for four filters at HRH ................................ ................................ ................................ ............................... 57 3 3 Removal efficiency, relative survival fraction, and quality factor based on viable removal efficiency of untreated filter and 12 hr treated DAC filter at two relative humidities in air filtration system ................................ ................................ ...................... 58 3 4 Comparison of other disinfection technology ................................ ................................ .... 59 4 1. Pressure drop (face velocity of 14.2 cm/s) of three types of filters treated with different concentrations of DAS suspension. ................................ ................................ ..... 75 5 1 Linear relationsh ip of the IE and SF of MS2 with temperature (T) ................................ ... 93 5 2. Pressure drop (face velocity of 5.3 cm/s) of three filters after microwave treatment at 375 W for 10 mins/cycle ................................ ................................ ................................ .... 93 6 1 Statistics in general factor Analysis of Variances ................................ ............................ 116 6 2 Virus susceptibility factor K (m 2 /J) for aerosol tran smission under different conditions ................................ ................................ ................................ ......................... 117 6 3 Virus susceptibility factors K (m 2 /J) from other studies ................................ .................. 117 7 1 Slope of least squares regression for N PFU as a function of particle size for different spray medium at three relative humidities. ................................ ................................ ...... 134 7 2 Slope of least squares regression for N RNA as a function of particle size for different spray medium at three relative humidities ................................ ................................ ....... 134

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9 LIST OF FIGURES Figure page 2 1. Schematic diagram of droplet loading system: ................................ ................................ .. 38 2 2 Loading density as a function: ................................ ................................ ........................... 39 2 3. CVs for Q T Q and S T S as a function of turntable speed ................................ .............. 40 2 4 S canning electron microscopy image s : ................................ ................................ .............. 41 2 5. Size distribution of droplets generated by ultrasonic nebulizer at five flow ra tes: ............ 42 2 6. The number and mass based particle size distributions of generated droplets and loaded droplets at 2 L pm ................................ ................................ ................................ .... 43 2 7. Recovery of viable MS2 as a function of extraction time for three FFRs ......................... 43 3 1 Experimental set up for the removal efficiency of the test filter in water f iltration .......... 60 3 2. Experimental set up: ................................ ................................ ................................ .......... 61 3 3. The number based particle size distribution of aerosols entering the filter at room temperature and low relative humidity ................................ ................................ .............. 62 3 4. Physical removal efficiency of four different filters as a function of particle size at room temperature and low relative humidit y ................................ ................................ ..... 62 3 5 Particle size distribution of the MS2 virus titer of 10 9 PFU/mL ................................ ........ 63 3 6. SEM images of the 12 hr treated DAC filter w ith collected E. coli before and after extraction ................................ ................................ ................................ ............................ 64 3 7. FT IR spectra of 12 hr treated DAC filter when untreated filter was used as background ................................ ................................ ................................ ......................... 65 3 8. SEM images: ................................ ................................ ................................ ...................... 66 4 1 Schematic diagram of the experimental set u p: ................................ ................................ 76 4 2 SEM images: ................................ ................................ ................................ ...................... 77 4 3 Performance of filters treated with different concentrations of DAS suspension: ............ 78 4 4 Relative survivability of MS2 viruses on filters treated with different concentrations of DAS suspension ................................ ................................ ................................ ............. 79 5 1 The experimental set up for microwave irradiation assisted f iltration. ............................. 94

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10 5 2 Thermal stability for three filters. ................................ ................................ ...................... 95 5 3 Temperature of the filters as a function of microwave applicat ion time at three different microwave power levels ................................ ................................ ...................... 96 5 4 Log inactivation efficiency and log survival fraction: ................................ ....................... 97 5 5 Mi crowave inactivation performance: ................................ ................................ ............... 98 5 6 Temperature of microwave and conventional ovens as a function of application time ..... 99 5 7 SEM images: ................................ ................................ ................................ .................... 100 5 8 Log inactivation efficiency by microwave irradiation assisted filtration system and and Log survival fraction on filter surface as a function of microwave power level: ..... 101 6 1. Schematic diagrams: ................................ ................................ ................................ ....... 118 6 2. Log inactivation efficiency by UV exposure at HRH for droplet and aerosol transm ission mode as a function of UV irradiation time in different nebulizer media .... 119 6 3. The SEM images of the filter contaminated with viruses aerosolized: ........................... 120 6 4. Log IE after virus loading and UV exposure at HRH for aerosol transmission mode as a function of UV irradiation time ................................ ................................ ................ 121 6 5. Log natural decay and inac tivation efficiency as a function of relative humidity during both loading and UV inactivation: ................................ ................................ ....... 122 7 1 Schematic diagram of the experimental set up: ................................ ............................... 1 35 7 2 Collection efficiency of BioSampler as a function of particle diameter with a sampling flow rate s of 4.5 and 12.5 Lpm. ................................ ................................ ....... 136 7 3 Particle size distribution of MS2 aerosols generated with DI water, beef extract, and artificial saliva at three relative humidities ................................ ................................ ...... 137 7 4 Particle size distribution of number ( solid) and mass (empty) based MS2 aerosols obtained from monitoring the SMPS and infectious viruses (cross) through plaque assay ................................ ................................ ................................ ..................... 138 7 5 The infectious MS2 per particle generated in DI water as a function of particle size at three relative humidities. Dash line represents the theoretical PFU per particle Error bar indicates the standard deviation of triplicate test ................................ ............ 138 7 6 The MS2 RNA per particle generated in DI water as a function of particle size at three relative humidities. Dash line represents the theoretical PFU per particle. Error bar indicates the standard deviation of triplicate test ................................ ...................... 139

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11 7 7. Stability factors of MS2 as a function of diameter at three relative humidities : ............. 140

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12 LIST OF A CRONYMS ABI Applied Biosystem ANOVA Analysis of Variance APS A erodynam ic P article S izer AS Artificial Saliva ATCC American Type Culture Collection BE Beef Extract BSL Biosafety Level CCF Coarse pore Cellulose Filter CDC Center for Disease Control and Prevention CF Cellulose Filter CFU Colony Forming Unit CMD C ount M edian D iameter CPC Condensation Particle Counter CV C oefficients of V ariation DAC Dialdehyde Cellulose DAS Dialdehyde Starch DI Deionized DLS Dynamic Light Scattering DMA Differential Mobility Analyzer DNA Deoxyribonucleic Acid E. coli Escherich ia coli ELISA Enzyme L inked I mmunosorbent A ssay

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13 ESP Electrostatic Precipitator FCF Fine pore Cellulose Filter FDA Food and Drug Administration FFR Filtering Facepiece Respirators FT IR Fourier Transform InfraRed HEPA High Efficiency Particulate Air HRH High Relative Humidity HVAC Heating, Ventilating, and Air Conditioning IE Inactivation Efficiency LD Lethal Dose LRH Low Relative Humidity MMD M ass M edian D iameters MPPS Most Penetrating Particle Size MRH Medium Relative Humidity MS2 MS2 bacte riophage NIOSH National Institute for Occupational Safety and Health PBS Phosphate Buffer Saline P CR P olymerase C hain R eaction PF Polypropylene Filter PFU Plaque Forming Unit PSD Particle Size Distribution qPCR Quantitative P olymerase C hain R eaction Q F Quality Factor

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14 Q T Q Quarter to Quarter RH Relative Humidity RNA Ribonucleic Acid RS Relative Survival fraction RT Room Temperature SARS Severe Acute Respiratory Syndrome SEM Scanning Electron Microscopy SF Survival Fraction SMPS Scanning Mob ility Particle Sizer STDA Simultaneous Differential Thermal Analysis S T S Sample to Sample TSB Tryptone Soy Broth TGA ThermoGravimetric Analysis UVGI U ltraviolet G ermicidal I rradiation VRE Viable Removal Efficiency WHO World Health Organization

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPROVING PROTECTION AGAINST VIRAL AEROSOLS THROUGH DEVELOPMENT OF NOV EL DECONTAMINATION METHODS AND CHARACTERIZATION OF VIRAL AEROSOL By Myung Heui Woo May 2012 Chair: Chang Yu Wu Major: Environmental Engineering Sciences Although respirators and filters are designed to prevent the spread of pathogenic aerosols, a stock pile shortage is anticipated during the next flu pandemic. Contact transfer and reaerosolization of collected microbes from used respirators are also a concern. An option to address these potential problems is to decontaminate used respirators/filters for reuse. In this research, a droplet/aerosol loading chamber was built and applied for decontamination testing for fair compar ison of the performance of different decontamination techniques Different decontamination techniques including antimicrobial chemic al agent s mic rowave irradiation, ultraviolet irradiation, were incorporated in t o the filtration system and evaluated. As biocidal filter dialdehyde cellulose / starch filter s were investigated for their inactivation efficacy. Both filter s with sufficient m oisture content had a higher removal efficiency, lower pressure drop, and better disinfection capability, which are all important attributes for practical biocidal applications For m icrowave assisted filtration system, temperature was identified to be a k ey factor. Relative humidity wa s another pivotal parameter for viability of viruses at warm to hot wa ter temperatures, but it bec ame insignificant at temperatures above 90 C As environmental conditions greatly affect the viability of microorganism, the e ffect of relative

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16 humidity and spray medium on UV inactivation w as examined. Absorption of UV by h igh water content and shielding of viruses near the center of the aggregate are considered responsible for lower inactivation Across different spray media, i nactivation efficiencie s in artificial saliva (AS) and in beef extract (BE) were much lower than in deionized water for both aerosol and droplet transmission indicating that solids present in AS and BE exhibited a protective effect. For particles sprayed in a protective medium, relative humidity wa s not a significant parameter The distribution of infectious MS2 aerosols follow ed volume based size distribution for pure viral aerosol whereas these of infectious MS2 generated with solid contents follow ed lo wer dimension s Aggregation by MS2 itself and encasement by inert salts yielded higher stability factor because of shielding effect and reduction of the air/water interface w hereas the soluble salts resulted in adverse effect. The knowledge and technologie s developed in this study can better protect the general publi c as well as healthcare facilities against viral aerosols.

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17 CHAPTER 1 INTRODUCTION Biological Threat r ecent years. During the 20 th century, three major influenza pandemics occurred from a major genetic change in the influenza strain: the Spanish flu in 1918 (20 100 million deaths), the Asian flu in 1956 (2 million deaths), and the Hong Kong flu in 1968 (1 million deaths). The 2002 and 2003 Se vere Acute Respiratory Syndrome (SARS), caused over 8000 cases and 700 deaths. The recent swine flu outbreak, due to a new strain of H1N1 influenza A, has caused illness in over 70 countries and resulted in at least 14, 000 deaths worldwide as of January 2010 (ECDC, 2010). On June 11 st 2009, World Health Organization (WHO) raised the pandemic alert level to Phase 6 awareness and concern of viruses of low infectious dose viruses and spread of airborne pathogens. Viral A erosol Bioaerosols are airborne particles with biological origins such as non viable pollen, and viable fungi, bacteria, and viruses (Burge, 1990). The adverse heal th effect of bioaerosols depends on several factors, such as microorganism type and dose. Among them, virus is the smallest one in size ( Prescott et al., 2006 ). Although the size of a single virion is smal l (20 nm 300 nm), viruses in nature exist in a wide range of sizes because of aggregation of several viruses, attachment of viruses onto other material, or encasement of viruses by droplets of respiratory secretions One important concern of the aggregate s is the adverse health effect imposed by the deposition in the respiratory system. Inhal ed particles can deposit in various respiratory regions. After they are deposited, the aggregates may disperse into numerous individual virions. More

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18 than 400 differen t viruses with different lethal doses (LDs) result in human diseases such as rubella, influenza, measles, mumps, smallpox, and pneumonia, which involve the respiratory system either directly or indirectly (Prescott et al., 2003). The aggregation, attachmen t, and encasement of viruses also facilitate resistance to environmental stresses, such as heat, dryness, toxic gases, and ultraviolet (UV) light because of shielding effect ( Kowalski & Bahnfleth, 2007 ) Although the aggregation state of virus with other m aterials, infectivity of viruses in aggregates under different environmental stress are key parameters to assess the health risk, very limited research has been carried out with respect to this aspect. Transmission M ode The understanding of the transmissio n modes of viral aerosol is critical to the protection of the public against major airborne pathogen pandemics. Effective prevention and treatment of infectious viral aerosols (i.e. vaccination and respiratory protection) also require specific information on the transmission mode. For the spread of infectious viruses, there are three critical transmission modes (CDRF, 2006): (1) Droplet transmission mode: Droplet transmission results from infected individuals generating droplets containing microorganisms by coughing, sneezing, singing, and talking (2) Contact transmission mode: Contact transmission includes a direct body to body contact and an indirect contact through a contaminated object (e.g., needle and towel). This mode frequently occurs in a healthcar e facility and ( 3) Aerosol transmission mode: Aerosol transmission includes the dispersion of droplet nuclei, which remains in air after evaporation of droplet, and dust particles containing the microorganism.

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19 Filtration Filtration is one of the most com monly used methods for collecting bioaerosols because of low cost and simple structure (Hinds, 1999). Aerosol filtration has been widely applied in various applications such as respiratory protection, air purification, and clean rooms (Lin et al., 2003). H igh efficiency particulate air (HEPA) filter is defined as one having at least 99.97% filtration efficiency for 0.3 MPPS (Hinds, 1999) Filtration efficiency is determined by several mechan isms such as interception, impaction, diffusion, gravity, and electrostatic force, depending on fiber density, diameter, filter thickness, and other factors (Hinds, 1999). The filtration efficiency at the MPPS is mainly determined by three mechanisms (Hin ds, 1999): (1) interce ption, where particles follow ing an airstream flow line come within one radius of a fiber and adhere to it; (2) impaction, where larger particles are unable to follow the curving contours of airstreams around th e fiber and are forced to strike on the fiber; and ( 3) diffusion, which is a result of the collision of small particles with gas molecules, which are thereby impeded in their path through the filter. Heating, ventilating, and air conditioning (HVAC) systems with HEPA filters can effectively control and reduce airborne contaminants including bioaerosols based on the above mechanisms ; however, reaerosolizations of virus collected inside an HVAC system in hospitals and residential buildings can be problems with this control strategy Surgical masks approved by the Food and Drug Administration (FDA) and filtering facepiece respirators (FFRs) certified by the National Institute for Occupational Safety and Health (NIOSH) are intended to be worn by healthcare persons and the general publ ic during a pandemic event. Virus might be filtered by the fibers of a regular surgical mask through diffusion. Also, aggregated viruses might be captured by impaction and interception. However,

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20 using surgical masks and N95 FFRs as countermeasures for bioa erosols has not been demonstrated to provide a complete response: (1) Although using surgical masks and N95 FFRs for tuberculosis has been shown to meet CDC guidelines, the same is not true for viral agents such as the swine flu virus. (2) There is no expe an H1N1 scenario can be estimated. (3) The stockpile of FFRs will be exhausted in the event of a severe pandemic. CDC estimates that more than 90 million FFRs will be required for healthcare workers in the U S if a pandemic influenza event persists for 42 days (CDRF, 2006). Decontamination M ethods One possible approach to resolve insufficient supplies of FFRs is to decontaminate the FFRs by using disinfection agents/processes such as microwave irradiation, UV irradiation bleach solution, and peroxide and then reuse the decontaminated respirator. For this, no change of FFRs characteristics and no adverse health effect from the chemicals should occur. However, currently there exists no protocol for decontaminat ion test for this purpose because of various limitations. First of all, there is no standard test method for simulating bioaerosol contamination of the FFRs. Secondly, the unique properties of bioaerosols generated by respiratory secretions may affect the effectiveness of the decontamination process, and the proper operating conditions to obtain controlled and consistent properties are not known. Thus, methods that can produce representative human respiratory secretions and device for a consistent and contr olled delivery of aerosolized droplets containing viral agents need to be developed in order to properly evaluate techniques for decontamination. Once a standard protocol for decontamination test is established, different methods can be fairly evaluated. T here have been several inactivation methods that can be applied to decontaminate filters loaded with viral aerosol (Fisher et al. 2009; Brion et al. 1999; Lee et al.

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21 2009; Zhang et al. 2009), including microwave irradiation, UV irradiation and antimicrob ial chemicals. Microwaves are electromagnetic waves with wavelengths between 1 m and 1 mm, or frequencies between 300 MHz and 300 GHz (Jones et al., 2002). Microwave s used in microwave ovens generally operate at a frequency of 2.45 GHz corresponding to a wavelength of 12 cm and energy of 1.0210 5 eV. Microwave radiation is non ionizing but is sufficient to cause polar molecules such as water to vibrate, thereby resulting in friction, which produces heat. The use of microwave irradiation for killing microo rganisms through thermal and non thermal effects has been demonstrated in various studies in liquid media (Pellerin, 1994; Kiel et al., 2002; Awuah et al., 2005; Campanha et al., 2007). However, no study for decon tamination of virus in ai r has been done. C onsequently, it is necessary to evaluate the performance of microwave irradiation against viral agent for air filtration system. UV light has sufficient energy to be a practical anti microbial method. UV irradiation is now a recognized method for inactivat ing a wide variety of biological agents and in particular airborne microorganisms (Prescott, 2006). Recent increases in the incidence of airborne diseases such as tuberculosis have focused attention upon the use of UV. With a wavelength of 254 nm, UV light strikes the biological cells and the energy is specifically absorbed by adjacent thymine nucleotide bases in deoxyribonucleic acid (DNA), causing them to form covalent bonds with each other rather than forming hydrogen bonds with adenine based in the comp lementary DNA strain ( Perier et al 1992) Phyrimidine dimers in thymine base distort the shape of DNA and change the double helix structure. Phyrimidine dimers make it impossible for the cell to accurately transcribe or replicate its genetic material whi ch ultimately leads to the death of the cell ( Kowaiski et al., 2007; Prescott, 2006 ). UV intensity, exposure time, lamp placement, air

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22 movement patterns and the relative humidity of the air determine the effectiveness of UV. Most studies have focused on th e UV intensity and exposure time to increase the decontamination efficacy ( Chang et al., 1985; Rastogi et al., 2006; Duleba Majek, 2009 ). None of them has considered the important parameters (e.g., relative humidity nebulized media, and transmission mode) related to susceptibility of viral agent. Therefore, the investigation of the effects of these parameters on decontamination efficiency is important in determin ing the optimal conditions. Incorporating antimicrobial agents such as aldehyde, phenolics, al cohols, halogens, heavy metals, and quaternary ammonium compounds into air filters are common methods to inactivate viruses. Among them, aldehydes such as formaldehyde and gluta r aldehyde are highly reactive molecules which combine with protein and nucleic acids by cross linking and alkylation. Recently, dialdehyde starch by glycol cleavage oxidation of starch through periodate reaction was shown to have antimicrobial effect with advantages such as low toxicity and low cost compared to gluta ra ldehyde (Hou et al. 2008; Para et al. 2004). Song (2008) demonstrated that dialdehyde polysaccharides including dialdehyde starch and dialdehyde cellulose synthesized by periodate oxidation act as biocides in aqueous suspension and by surface contact. However, its incorp oration into air filtration media has not been explored. As a consequence, evaluation of the decontamination performance of dialdehyde cellulose filter against airborne viral agent to determine the feasibility of its use in a wide range of app l ication is n ecessary Research Objectives The ultimate goal of this doctoral research was to improve protection against viral aerosol through development assessment and comparison of n ovel decontamination technologies T he first objective wa s to develop a method for consistent and controlled delivery of droplets containing viral agents onto surface that would allow fair comparison of different contamination

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23 technologies The second and third objectives were to evaluate the performance of dialdehyde cellulose filter an d dialdehyde starch filter against viral aerosol, respectively. The fourth objective wa s to evaluate microwave irradiation assisted filtration for capture and inactivation of viral aerosols for collective protection The fifth objective wa s to i nvestigat e the effects of relative humidities and nebulized media on UV decontamination of viral aerosols and viral droplets loaded filter for individual protection The sixth objective was to investigate the stability of MS2 virus by comparing infectious virus to t otal virus under different environmental conditions The comprehensive study about development and evaluation of inac tivation technologies based on the characteristics of vir al aerosol will lead to mitiga tion of the shortage problem of respirator stockpil e and to provide novel means for inactivating airborne biological agents, subsequently to alleviate the threat of disease transmission by viral aerosol. If successfully accomplished, the knowledge learned and technologies developed can better protect the g eneral publi c as well as healthcare facilit ies against viral agents.

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24 CHAPTER 2 METHOD FOR CONTAMINA TION OF FILTERING FA CEPIECE RESPIRATORS BY DEPOSITION OF MS2 VI RAL AEROSOLS Background ck) and spread of airborne pathogens (e.g., Severe Acute Respiratory Syndrome (SARS) and avian flu (H5N1)) through the aerosol route has increased greatly in recent years (Tellier, 2006). For example, in 2002 and 2003 SARS caused over 8000 illnesses and 70 0 deaths and there is still no adequate treatment ( Yang et al., 2007). The recent swine flu outbreak due to a new strain of H1N1 influenza A has caused illness in over 70 countries and resulted in at least 6000 deaths worldwide as of October 2009 (ECDC, 20 09). On June 11, 2009, the World Health Organization (WHO) raised the pandemic alert level to Phase 6, indicating the onset of a global pandemic (CDC, 2009). One effective method for protection against airborne pathogens during pandemic spread through dro plet and aerosol transmission is to wear a filtering facepiece respirator (FFR) certified by National Institute for Occupational Safety and Health (NIOSH). This approach cons iderably decreases the incidence and severity of infection. The Center for Disease Control and Prevention (CDC) has issued guidelines about the use of face masks and respirators to protect against H1N1 transmission in healthcare facilities (CDC, 2009). However, using medical masks and N95 FFRs as countermeasures for bioaerosols has not been demonstrated to provide a complete response: (1) Although using surgical masks and N95 FFRs for tuberculosis has been shown to meet CDC guidelines, the same is not true for viral agents such as the swine flu virus. (2) There is no experimental basis u Reprinted with permission from Woo, M H., Hsu Y M Wu C Y, Heimbuch B Wander J. (2010). Method for contamination of filtering facepiece respirators by deposition of MS2 viral aerosol. J ournal of Aerosol Sci ence 41, 9 44 9 52.

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25 estimated. (3) The stockpile of FFRs will be exhausted in the event of a severe pandemic. CDC estimates that more than 90 million FFRs will be required for healthcare workers in the US if a pandemic influenza event persists for 42 days (CDRF, 2006). One possible approach to resolve insufficient supplies of FFRs is to decontaminate the FFRs by applying disinfection agents/processes such as microwave irradiation, ultraviolet germicidal irradiation (UVG I), bleach solution, peroxides, etc, and then reusing the decontaminated respirator. To qualify a method to decontaminate an FFR for reuse, one must provide a statistically robust demonstration that the technologies applied do not alter the mechanical prop erties of the FFR, do not leave any toxic byproduct on the FFR, and achieve at least four log virucidal efficacy on the materials of construction of the FFR. However, no protocol has been reported for such decontamination testing, a consequence of two main limitations. First, no standard test method has been reported for simulating bioaerosol contamination of the FFRs. Second, the unique properties of bioaerosols generated by respiratory secretions can be expected to affect the efficacy of the decontaminati on process, and the window of operating conditions affording controlled and consistent properties is not known. Therefore, development and validation of methods that are representative of human respiratory secretions is a necessary condition before one can realistically evaluate techniques for decontamination. Influenza is commonly thought to be transmitted by three mechanisms (droplet, contact, and aerosol (droplet nuclei)) (CDRF, 2006). Some diseases (e.g., tuberculosis ) are known to be mainly transmitte d by the droplet nuclei route whereas droplet transmission is considered by many to be the dominant route for some other diseases (e.g., mumps ), although the actual routes a re still being debated (Fiege l et al. 2006 ; Yang et al. 2007; Tellier 2006 ) Salgado et al. (2002) suggested different roles in influenza between droplet and aerosol transmission. Influenza is

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26 mainly spread through droplet transmission by coughing and sneezing from infectious people while aerosol transmission is important for long distance and sporadic infection. An infected human can be a source of large droplets generated by coug hing and sneezing. During airborne transmission these droplets will shrink in size with the consequence that both droplets and droplet nuclei contact surfaces. Although many researchers have examined the droplet size generated from humans, the actual size is not clear. Yang et al. ( 2007) reported that most droplets from coughing, sneezing, and talking have diameter between 1 and 20 m and these droplets may contract depending on the humidity and medium generated. Viruses in these droplets can aggregate with each other or be encased by the saliva component both enhancing persistence of viability. Meanwhile, viability of viruses in s aliva can be atten uated by enzyme action (Dia & Marek, 2002). Therefore, it is important that the transmission medium be factored into the design of the test method. The focus of this study wa s to develop a method for reproducibly applying fixed amounts o f representative viral particles generated from droplets/aerosols onto FFRs for decontamination testing. This study had specific objectives : (1) to build a droplet/ aerosol chamber system that generates droplets/aerosols containing viruses to emulate those from coughing and sneezing ( 2 ) to deliver the droplets and resulting aerosol onto specimens of six commercially available FFRs, and ( 3) to demonstrate uniformity of deposition within a sample and across independent samples by achieving quarter to quarter (Q T Q) and sample to sample (S T S) coefficients of variations (CVs) of less than 20% and 40%, respectively. While the focus of this study was FFRs and a biosafety level (BSL) I virus, the system was designed to be used with BSL II microorganisms which re quire advanced containment. This fact limited the overall size of the unit due to the

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27 requirement for secondary containment of the test system. The system also has utility outside of FFRs and could be used to load any surface (e.g., surgical scalpel or glo ve). Material s and Methods Virus and Nebulization F luid P repar a tion MS2 bacteriophage (MS2; ATCC 15597 as the first challenging bioaerosol because it requires only a BSL I facility. MS2 has a nonenveloped, icosahedral capsid with a nominal diam eter of 27.5 nm (Prescott Harley, & Klein, 2006 ; Valegard Lijas, Fridborh, &Unge 1990 ). M S2 infects only male E scherichia col i ( E. col i ) and is commonly used as a non pathogenic surrogate for human pathogenic viruses (e.g., poliovirus influenza A, and rhinovirus ) because of its similarity in resistance to antimicrobial agents and ease of prep aration and assay (Brion & Silverstein, 1999 ; Aranha Creado & Brandwein, 1999 ; Fisher et al., 2009 ). Freeze dried MS2 was suspended in deionized ( DI ) water to a titer of approximately 10 10 10 1 1 plaque forming units (P FU s ) per mL and stored at 4 C. Arti ficial saliva was used as the nebulization fluid to emulate droplets generated by coughing and sneezing Saliva is a very dilute fluid composed of more than 97% water, plus electrolytes, proteins, and enzymes (Diaz & Marek, 2002). Varieties of inorganic io ns maintain osmotic balance and offer buffering (Humphrey & Williamson 2001; Diaz Arnold & Mark 2002; Dodd s et al. 2005). The compounds and their corresponding amounts of artificial saliva are listed in Table 2 1 ( Vee r man et al. 1996; Wong & Sissions 2001; Aps & Martens 2005; Edward et al., 2004) Mucin from porcine stomach (Sigma Aldrich, M1778) was chosen as the representative mucus stimulant (Vingerhoeds et al., 2005). Test M aterial Six different models of FFRs approved by NIOSH were employed in t his study. Three of those models were also approved by the Food and Drug Administration (FDA) as surgical

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28 devices. Each type of FFR has different characteristics, such as number of layers hydrophili city, and physical shape. Prior to FFR testing, 110 mm di ameter discs of flat glass fiber filter (Gelman Science, 61630) were used to determine workable operating conditions of virus concentration, flowrate, and loading time. Droplet/ A erosol L oading S ystem A droplet /aerosol loading system was custom built for th is study with the following requirements: (1) the width and height are less than 120 cm so that it can be placed in a biosafety cabinet; (2) parts can be easily disassembled for sterilization; (3) droplets/aerosols can be distributed uniformly onto substra tes; (4) the droplet size distribution is consistent; (5) environmental conditions that can affect the droplet size, such as relative humidity (RH) and temperature, can be controlled. The schematic design of the l oading system is shown in Fig ure 2 1 The s ystem consists of a chamber body, an ultrasonic generator (241T, Son ae r Farmingdale, NY) for producing the droplets, a bubbler for generating moisture, compressed cylinder air for controlling RH and diluting virus concentrations, an RH meter for measurin g humidity, a six port manifold for distributing the aerosols, a thermometer for measuring temperature, and six supports to hold the flexible form FFRs during loading ( Baron et al., 2008 ; Feather & Chen 2003 ; Fisher et al. 2009 ). This system can also inc lude a charge neutralizer (Model 3012, TSI Inc., Shoreview, MN). The chamber body was fabricated from stainless steel sheet with welded seams to withstand the high temperature for sterilization The turntable and six perforated sample plates were employed to increase uniformity of deposition The particle size distributions (PSDs) were measured by an aerodynamic particle sizer (APS ; Model 3321, TSI Inc., Shoreview, MN ) through a port on the side of the chamber Before building the droplet loading chamber, a n experiment was conducted to verify the uniform deposition of aerosols using a small chamber, presented in Appendix A.

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29 Chamber O peration and D etermination of O perating C onditions Before and after experiments, the chamber was decontaminated by wiping the i nterior of the chamber with isopropyl alcohol then allowing the chamber to set for 30 minutes. Six samples were placed onto the supports on the turntable using sterile forcep s Theoretically, a titer of around 10 7 PFU/mL in the ultrasonic nebulizer with 5 min loading time should provide sufficient loading density (> 10 3 PFU/ cm 2 ). The titer was prepared by adding 0. 3 mL virus stock suspension into 3 0 mL artificial saliva. T he droplets from the ultrasonic nebulizer after passing the distributor entered the cha mber through six inlets. The size of droplets generated and loaded can be affected by the frequency of the ultrasonic generator and by environmental conditions such as RH and temperature. For this study, the frequency of the generator was 2.4 MHz and the e nvironmental conditions were 2 0 2 C and 35 5%. Low RH was chosen because the survivability of MS2 is high under this condition. After loading, the residual droplets were allowed to clear for 5 mins, and the FFR samples were taken out for extraction and as say. Various operating parameters were evaluated to determine the conditions that would provide desired droplet characteristics, including loading time (1 30 mins), virus titer (10 7 10 8 PFU/mL), turntable speed (0 3 rpm), airflow rate (1 5 Lpm), and mucin concentration (0.3 0.9%). The loading density was controlled by adjusting the loading time and the titer of the virus suspension. To evaluate how viability of the virus is influenced by the ultrasonic process, bioaerosols produced at different times were c ollected by a Biosampler and their viability was compared. The turntable speed was varied to determine its relationship with uniformity. Flow rate and mucin loading were also varied to investigate their effects on the consistency of delivered droplets. Thr ee runs were carried out for each set of conditions.

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30 After loading with virus, each filter sample was cut into four equal quarters. Each quarter was immersed in 25 mL of extraction medium in a 50 mL conical tube. A 0.25 M glycine solution was applied to e xtract the MS2 from the quarter sample with agitation by a wrist action shaker (Model 75, Burrell Scientific, Pittsburgh, Pa.) at a 10 angle for 15 mins to analyze the loading density (these conditions showed the best extraction efficiency in preliminary testing). The extracted solution was assayed by using the single layer method (EPA, 1984) to determine the loading density according to Eq uation 2 1 with the assumption of 100% extraction efficiency: LD (2 1) where LD is the loading density, V 1 is the volume of extraction solution, V 2 is the volume of sample, d is the diameter of the filter, and n is the number of dilutions (Lee et al., 2009). A single layer bioassay was used to enumerate the infectious viruses with a host of Escher ichia coli ( E. coli ; ATCC, 15597). The freeze dried E. coli was suspended in 1X PBS, and isolated into a solidified hard agar plate (1.5% agar) with a sterilized loop, and then incubated at 37 o C overnight. The single colony from the plate was transferred to the tryptone soy broth (TSB ) 271 for E. coli growth at 37 o C overnight. The MS2 media 271 (100 mL) was inoculated with 0.3 mL of the E. coli culture from TSB 271 and then incubated at 37 o C for 3 hrs. The TSB 271 and culture medium 271 were prepared fol lowing the American Type Culture Collection (ATCC) procedure for MS2 assay. One millimeter of MS2 sample and 3 hrs incubated E. coli host were added to the sterile conical blue tube containing 9 mL of soft agar (0.5% agar) in a water bath between 40 o C an d 50 o C. To get the countable range of 30 300 PFU/mL, the serially diluted MS2 samples were used. The mixture was shaken thoroughly and then poured into a petri dish. After the agar hardened, the plate was inverted and placed in an incubator at 37 o C

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31 overn ight. The plagues on the plate were enumerated and the titer of the sample was determined after multiplying the dilution factor to the plaque count. To visualize the particle loading, scanning electron microscopy (SEM, JEOL JSM 6330F, JEOL Inc.) of the fil ter before and after loading particles. Statistical A nalysis The Q T Q and S T S CVs of loading density were obtained to evaluate the uniformity. R2 8.1 software (CRAN) and Microsoft Excel were used to calculate one way analysis of variance (ANOVA) and CV, respectively. Results and Discussion D etermination of O perati ng C onditions The impact of ultrasonic nebulization on viability of virus in the nebulizer reservoir was investigated by measuring the viable counts over time. The results present no signifi cant difference in virus viability between 0 and 30 mins ( p =0.10) (data not shown) Apparently, the heat shock from ultrasonic vibration did not cause damage to the MS2 in the reservoir during droplet generation. T o determine the effect on virus of ultraso nication during droplet generation, viability of the viruses collected in the BioSampler after 5 and 10 mins of generation was examined. The theoretical concentration in the BioSampler after 5 mins of nebulization is 3 10 5 PFU/mL when the virus titer in th e reservoir is 1.0 10 7 PFU/mL. The 5 min time weighted (0 5 and 5 10 mins) average concentration of collected viruses in the BioSampler was around 3.2 10 5 PFU/mL, which is similar to the theoretical value. As demonstrated, the ultrasonic nebulizer can be u sed to produce droplets containing MS2 virus without adverse effects on viability.

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32 Fig ure 2 2 A displays the virus loading density as a function of loading time and Fig ure 2 2 B shows the loading density as a function of the viral titer As shown, the loadin g densi t y had a linear relationship with time and with virus titer in the nebulization medium as expected based on the initial viability tests. The results show that these two parameters can be adjusted to acquire a desired loading density. It should be n oted that i n determining the loading density the extracted fraction was assumed to be 1 ; however, different types of FFRs will have different values that depend on the material property and structure. The uniformity tests were conducted with the turntable at various speeds because Marple & Rubow (1983) observed increased uniformity of CV from 4.3% to 1.5% when they rotated their aerosol chamber at 0.56 rpm. Fig ure 2 3 shows the CVs from three runs as a function of turntable speed. The variation of the CVs w as somewhat larger than Marple & Rubow (1983) reported due to variability working with a viable system (MS2) vs. a non viable system (PSL and dust). The flow rate (2 Lpm) of this work is much lower than the rate (100 Lpm) used by Marple & Rubow (1983), and delivers a distribution of droplets that is sufficiently uniform ( CV < 20%) even without the turntable, which appears to provide a moderate decrease in CV with increasing rotation rate. The difference among the six positions was not statistically signifi cant ( p = 0.73) for flat sheet glass fiber filters. The reason for this uniformity is likely because settling is the dominant mechanism for large droplets in our system and a six port distributor delivered the bioaerosols A straight fog stream was observe d through the front window during the loading. Therefore, the turntable speed was not an important parameter to meet the uniformity criteria when the flatsheet filter was employed The deposition of the particles on FFRs after loading for

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33 1 min was also co nfirmed by SEM. As shown in Figure 2 4, particles were randomly distributed on fiber surface without any specific pattern. The criterion for minimum loading density 10 3 PFU/ cm 2 was achieved for all conditions tested and could be easily increased if needed. Based on these results, the operating conditions to be discussed later were chosen to be 5 min loading time at a titer of 10 7 PFU/mL and 2 rpm turntable speed. Dropl et S ize D istribution Fig ure 2 5 shows the size distribution of droplets generated by the u ltrasonic nebulizer at different flow rates. The size distribution s of droplets generated at 1 3 L pm were similar, with count median diameters (C MD s) and mass median diameters ( MMD s) of 3.5 m and 10 m, respectively. The droplet size distribution s at 4 and 5 Lpm were slightly shifted to a smaller diameter. Bimodal distribution was observed in the mass based size distribution, with modes at 4 6 m and over 20 m. The theoretical CMD from the ultrasonic nebulizer can be determined from Eq uation 2 2 ( Lang 1962 ) : d p (CMD) = 0.73 ( 2 2) where d p is the nebulized droplet size, is the surface tension of the liquid, is the density of the liquid, and f is the frequency of th e nebulizer As shown, the droplet size is independent of the flow rate The gentle airflow within 1 3 Lpm just carries the aerosol away from the liquid surface. However, at a higher flow rate, the larger volume of dry dilution air promotes evaporation and therefore results in a smaller droplet size. To generate droplets of other sizes, the frequency can be adjusted. For example, droplets are expected to be 12.1 and 6.6 times larger, respectively, when lower frequencies of 60 kHz or 150 kHz are used instead of 2.4 MHz ( Lang 1962 )

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34 The mucin concentration also plays an important role in determining the initial droplet size because it affects surface tension and density of the artificial saliva used. Mucin at 0.3% was chosen for the artificial saliva in this study to match the protein content in human saliva. I ncreasing the mucin concentration threefold would reduce the median size to 40% of its original size because of the decrease in surface tension In summary, the droplet size can be controlled by adjusti ng the composition of the spray medium the frequency of the ultrasonic generator and the flow rate The droplet size decreases from the point of generation at the ultrasonic nebulizer all the way to the filter surface due to evaporation, and the size dep osited depends on the environmental conditions (i.e., temperature and RH). The flowrates of the ultrasonic generator, dilution air, and temperature of the bubbler can be used to control RH, and heating tape (part (b) in Fig ure 2 1) can be used to adjust th e temperature. Fig ure 2 6 displays the droplet size distribution generated and loaded at 2 L pm through the aerosol generator plus 3 L pm dry air to provide 35% RH. For this condition, t he cylinder air without a bubbler was applied to achieve the low RH cond ition. The MMDs for droplets generated and loaded were 9.2 and 3.2 respectively, with corresponding C MD s of 3.4 and 1.8 which are similar to the droplet size reported in the literature ( Yang et al. 2007; Morawska et al 2009) to have been generated by humans The is considered the main mechanism acting in this droplets just reaching the FFR are almost completely evaporated. The size of a completely evaporated droplet can be c alculated according to Eq uation 2 3 : d p = d d (2 3)

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35 where d p is the diameter of the aerosol particle and Fv is the volume fraction of solid material in the suspension in the nebulizer. For 0.3% mucin, the volume fraction is 6 .0 0 10 2 so d p is calculated to be 1 9 d d theoretical value is incomplete evaporation. After running the experiment, we noted that the filter surface was slightly damp, which is consistent with th e above interpretation. Loading onto NIOSH certified FFRs The CVs for uniform deposition of droplets /aerosols onto substrates for six different FFRs were calculated by analyzing the infectivity of viruses extracted from the loaded filter (Table 2 2 ). The f lexible nature of the FFRs make s it difficult to achieve deposition on the same spot with the same shape each time. Therefore, a holding medium is necessary to achieve low CV values. Even for fixed form FFRs, some inherently cannot produce equal quarters b ecause the shape is not symmetric. Operational variation while cutting the sample (e.g., uneven quarters) can also contribute to larger CV values. Consequently, the CV for Q T Q was higher than that for S T S over all FFRs. It is possible to use circular a reas punched from a FFR so that the difference in shape will not influence the results. However, this was outside the scope of the study, which aimed to evaluate decontamination effectiveness using the entire FFR. Nevertheless, the average CVs for both Q T Q and S T S for all FFRs were lower than the criteria 20% and 40%, respectively Table 2 2 also displays the results of the loading densities As shown, all sets had sufficient quanti ty to meet the threshold criteri a Due to differences in surface properties, the loading density of different respirator models can be different even when the same operating conditions are applied. Fig ure 2 7 shows the extraction efficiency of three diff erent FFRs at various extraction times. Different layer structures and properties of the FFR s are responsible for the differences as discussed The shape

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36 of the FFR is another reason for differences in loading density The loading density reported in Table 2 2 was calculated based on projected area. The FFR having a duck bill shape showed a lower loading density owing to its different curvature. As shown, both factors can affect loading density Nevertheless, the loading density can be easily met in a controlled fashion using the specified conditions and can be conveniently increased by adjusting the loading time, virus titer, or both Summary A simple system for producing delivering and loading of consistent challenges of droplets /aerosols conta ining virus onto FFRs has been developed and assessed. The respective CVs for S T S and Q T Q for the six NIOSH certified FFRs tested were lower than 20% and 40%. The droplet size can be altered by tuning the frequency of the ultrasonic nebulizer by chang ing the composition of the dispersion aerosolized, and by adjusting the temperature and RH inside the chamber. Droplets emulating bioaerosols released during coughing and sneezing can be produced using specific conditions and the loading density can be ach ieved by controlling the loading time and the virus titer in the nebulization medium This system allows for development and validation of a standard method for loading bioaerosol challenges when different decontamination techniques are to be compared. It also has utility for loading surfaces to study fomite transmission and reaerosolization of particles from surfaces. It can be further applied to generate and load droplets and aerosols of different sizes and to load onto materials other than FFRs.

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37 Table 2 1 Composition of artificial saliva (Based on 979 mL of DI water) Chemical Species Amount Chemical Species Amount MgCl 2 7 H 2 O 0.04 g KSCN 0.19 g CaCl 2 H 2 O 0.13 g (NH 2 ) 2 CO 0.12 g NaHCO 3 0.42 g NaCl 0.88 g 0.2 M KH 2 PO 4 7.70 mL KCl 1.04 g 0.2 M K 2 HPO 4 12.30 mL Mucin 3.00 g NH 4 Cl 0.11 g DMEM 1mL DMEM : Dul Table 2 2 Loading density and CVs of Q T Q and S T S for 6 different FFRs (N = 3, Criteria of CV for Q T Q and S T S: 20% and 40% respectively ) Shape Type No Loading density (PFU/cm 2 ) CV for Q T Q (%) CV for S T S (%) Fixed P 1 2.3 10 3 0.3 10 3 12.07 2.74 9.05 2.22 P 2 2.9 10 3 0.2 10 3 10.92 2.09 5.89 0.68 S 3 1.0 10 3 0.1 10 3 15.41 6.89 10.12 3.75 P 4 2.6 10 3 0.2 10 3 18.04 2.97 6.94 3.26 Flexible (duckbill) S 5 1.2 10 3 0.1 10 3 13.70 1.59 9.17 3.86 S 6 1.8 10 3 0.2 10 3 13.19 7.19 10.27 1.71 N = 2, P :Particulate respirator, S :Surgical respirator

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38 Figure 2 1 Schematic diagram of droplet loading system: A) entire system B) distributor on top C) turntable D) air outlet at bottom ( a ) and b ) are needed only in certain conditions)

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39 Figure 2 2 Lo ading density as a function: A) loading time and B) virus titer in the nebulizer

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40 Figure 2 3 CVs for Q T Q and S T S as a function of turntable speed

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41 Figure 2 4 S canning electron micro scopy image s : A ) untreated FFR and B) treated FFR at 250x

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42 Figure 2 5 Size distribution of droplets generated by ultrasonic nebulizer at five flow rates: A) N umber and B) mass based

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43 Figure 2 6 The number and mass based particle size distributions of generated droplets and loaded droplets at 2 L pm through the aerosol generator plus 3 L pm dry air and 2 rpm turntable speed Figure 2 7 Recovery of viable M S2 as a function of extraction time for three FFRs

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44 CHAPTER 3 EVALUATION OF THE PE RFORMANCE OF DIALDEH YDE CELLULOSE FILTER S AGAINST AIRBORNE AND WATERBORNE BACTERIA AND VIRUSES Background The increasing concern of bioterrorism ( e.g. anthrax attack), the spread of waterborne pathogens ( e.g. typhoid fever) and airborne pathogens ( e.g. avian flu, Severe Acute Respiratory microorganisms and protection methods against them ( Prescott, 2006; CDC, 2008) Filtration is one of the most commonly applied methods to remove airborne and waterborne microorganisms because of its low cost, simple structure, and high efficiency (Hinds, 1999) Air filtration has been widely applied as resp iratory protection and air purification (Lin & Li, 2003 ) while water filtration has been applied for drinking water purification and waste water treatment (Metcalf, 2004) Although filtration is an efficient method to capture viable microorganisms as well as nonviable particles, it does not inhibit the survival of viable microorganisms collected on the filter, thus allowing the filter to be a fomite. The viable particles may grow on the filter and even reaserosolize from the filter through high air stream, causing adverse health effects. Therefore, inactivation treatment is necessary to decontaminate the loaded filters. Several disinfection technologies, including irradiation, dry heat, and chemical treatment have been studied to decontaminate loaded filte rs. Ultraviolet (UV) irradiation is used for air and water sterilization because UV C with wavelength of 254 nm breaks the molecular structure within deoxyribonucleic acid ( DNA ) or r ibonucleic acid ( RNA ) and makes thymine dimmers; however, it has a limitat ion of low penetration due to short wavelength (Kujunnzic et al., 2006) Microwave irradiation is also applied for wet/dry disinfection with the thermal effect by water Reprinted with permission from Woo, M H., Lee, J. H., Rho, S. G., Ulmer, K., Welch, J., Wu, C. Y., Song, L. & Baney, R. (2011) Evaluation of the p erformanc e of d i a ldehyde cellulose fi lter against airborne and waterborne bacteria and virus. Ind ustry & Eng ineering Chem istry Res earch 50, 11636 11643

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45 vibration and non thermal effects such as cell damage, membrane distortion, and change of enzyme activities ( Wu & Mao 2010) R ecently, flash infrared radiation disinfection has been introduced as fomite disinfection (Damit et al. 2010) However, all irradiation based methods require an additional energy source and a facility for the treatm ent. Besides radiation based methods, dry heat and chemical treatment have also been used ( Viscusi et al. 2009) High temperature can cause protein denature and chemical reaction can distort biomolecular functions. Vis cusi et al. (2009) showed the deconta mination effect of autoclave and 160 o C dry heat, 70% isopropyl alcohol, and soap water against MS2, but also reported the significant degradation of filtration efficiency after one time decontamination treatment. Therefore, a decontamination technology wi thout additional energy source and without causing filter degradation is necessary. Without the above limitations, biocidal filters have been approached to take advantage of combined disinfection and mechanical filtration. Halogen compounds are effective a ntimicrobial agents that can be incorporated to filters because of their ability to damage the capsid protein (Ratnesar Shumate et al., 2008). Ratnesar Shumate et al. (2008) r eported that iodine quaternary ammonium treated filter has biocidal effects agai nst Micrococcus luteus and E. coli and Lee at al. (2009) demonstrated that the iodine dislodged from the filter can inactivate MS2 for air filtration. Verdenelli et al. (2003) also demonstrated that chloride treated filters (cis 1 (3 chloroallyl) 3,5,7 tr iaza 1 azoniaadamanta ne chloride and octadecylmethylt rimethoxysilylpropyl ammonium chloride) have higher filtration efficiency, lower microbial colonization, and longer lifetime against 10 different bacteria and 6 different fungi for air filtration. Miller (2009) reported that perlite covalently bonded by quaternary a mine organosilane (3 trihydroxysilylpropyl dimethyloctadecyl ammonium c hloride) destroys pathogens ( E. coli ) by piercing the outer

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46 membrane during water filtration. However, with these types o f biocial filters there is a possibility of toxic chemical release chemical s to humans. First reported in 1964 aldehydes such as glutaldehyde have also been used as antimicrobial agents against bacteria, spores, fungi, and viruses (Mcdonnell & Russell, 19 99) Several studies demonstrated that aldehydes work through the following mechanisms: (1) strong interaction with outer cell layers in bacterial spores, (2) cross linking in protein, transport inhibition, and strong association with outer layers in both gram negative and positive bacteria, (3) interaction with cell wall in fungi, and (4) inhibition of DNA and RNA synthesis in viruses (Power & Russell, 1990; Bruck, 1991; Favero, 1991) However, the small molecular aldehyde is also toxic. In contrast, large r aldehyde compounds are less toxic. Recently, dialdehyde starch by glycol cleavage oxidation of starch through periodate reaction has been shown to have antimicrobial effect, low toxicity and low cost compared to gluta r ldehyde (Hou et al. 2008; Para et a l., 2004) Song (2008) demonstrated that dialdehyde polysaccharides including dialdehyde starch and dialdehyde cellulose synthesized by periodate oxidation act as biocides in aqueous suspension and by solid contact. However, its incorporation into air and water filtration media has not been explored. The objective of this study was to investigate the filtration efficiency of the dialdehyde cellulose filter synthesized by one step periodate oxidation. The inactivation performance of this filter against airbo rne and waterborne bacteria and viruses under different environmental conditions was evaluated. For water filtration the viable removal efficiency and the relative survival fraction of untreated and treated filters of different treatment times were invest igated by testing with both E. coli and MS2 bacteriophage. For air filtration the physical removal

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47 efficiency, viable removal efficiency, and relative survival fraction of filters under different relative humidities (RHs) were investigated. Material s and Methods Test F ilters The dialdehyde cellulose (DAC) filters were prepared by periodate oxidation of cellulose i.e., 4, 8, and 12 hrs) (Song, Cruz, Farrah, & Baney, 2009) The cellulose filters were immersed int o 100 mL of deionized (DI) water containing 0.2 M sodium periodate (NaIO 4 Fisher Chemical) with pH of 3 4. The reaction was performed in a shaker at 200 rpm of speed in the dark at 37 C for the varying treatment times. Subsequently, the filter was rinsed five times with DI water. The filter was then immersed in the same volume of DI water in a shaker at room temperature (RT) overnight. This filter was again washed several times to remove excess periodate. After rinsing out the residual periodate, the DAC filter was dried in a hood at RT for 24 hrs. As a control, an untreated cellulose filter was used. To ensure no residual periodate, the treated filter was tested with 2 mL of 0.5% (w/v) sodium metabisulfite aqueous solution and 1 mL of 0.1 N silver nitrate to observe if there was any color change resulting from the residue. To determine the change of chemical structure after treatment, Fourier Transform InfraRed (FT IR; Nicolet Magma 560) spectroscopy in the wave number range of 4000 500 cm 1 was used. Test Bacteria and Bacteriophages E. coli (ATCC 15597 ) and MS2 (ATCC 15597 were used as the challenging agents for water and air filtration. E. coli a gram negative rod shaped bacterium with an aerodynamic diameter 0.7 0.9 m, is commonly used as a repr esentative of vegetative cell. Freeze dried E. coli was suspended with 1% tryptone soy broth (TSB, BD 211705) to a titer of 10 11 10 12 colony forming units (CFUs)/mL. E. coli stock was prepared by inoculating the E .coli

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48 onto a nutrient agar (Becton, Dickin son and Company) plate and then transferring the inocula te to a slant. This slant was incubated for 24 hrs, and E. coli suspension extracted from the slant through vortexing with 1X phosphate buffer saline (PBS) was used as the bacteria stock. 10 The final titer of E. coli was around 10 8 10 9 CFU/mL after serial dilution. MS2 is a single strand RNA, unenveloped, and icosahedra shaped virus with a single size diameter of 27.5 nm. Because of similar physical characteristics, MS2 has been used as a surrogate for human pathogenic RNA viruses such as poliovirus and rotavirus (Pre scott et al., 2006; Woo et al. 2010) Freeze dried MS2 was suspended with 1% TSB to a titer of 10 11 10 12 plaque forming units (PFUs ) /mL as the virus stock suspension and stored at 4 C. The stock was serially diluted to 10 8 10 9 PFU/mL by sterile DI water before being used for the experiment. Water F iltration The water filtration system was comprised of a vacuum based filter (Model XX11047 00 Sterifil Aseptic System, Millipore Co.) loaded wi th either an untreated cellulose or a treated DAC filter, as shown in Fig ure 3 1. Ten mL of bacterial or virus suspension with a titer of 10 8 10 9 CFU (or PFU)/mL were dripped through the filter at 0.5 Lpm under vacuum for 1 min (Herrera et al. 2004) To d emonstrate the applicability for real life scenario, Suwannee river assayed to investigate their viable titers using suitable dilution to an adequate count ( i.e 30 300 CFU or PFU) by the single layer method (EPA, 1984) For enumeration of bacteria and virus, 9 mL of agar medium 271 was prepared in a 15 mL conical tube (Falcon, Becton, Dickinson and Company) and placed in a water bath at 50 o C (Fisher et al. 200 9) For E. coli 1 mL of diluted E. coli suspension was added to the tube. For MS2, 1 mL of diluted MS2 suspension and 1 mL of log phase E. coli prepared in medium 271 were added to the tube (Fisher et al. 2009) The agar

PAGE 49

49 containing E. coli or MS2 was mix ed by swirling and then the mixture was poured into the Petri dish. The agar solidified at room temperature and then the dish was put into the incubator at 37 o C for 24 hrs before counting. Scanning electron microscopy (SEM, JEOL JSM 6330F, JEOL Inc.) and dynamic light scattering (DLS, Nanotrac NPA252, Microtrac Inc.) combined with Zetasizer (Malvern Instruments) were used to confirm the aggregation of the viruses. Air F iltration The experimental set up for air filtration is displayed in Fig ure 3 2 with two compartments for viable removal efficiency and physical removal efficiency measurements. Compressed air was passed through two rotameters. One allowed 7 Lpm of dry air to pass though a six jet Collison nebulizer (Model CN25, BGI Inc.) to aerosolize a bact erial suspension with a titer of 10 7 10 8 CFU/mL, and the other allowed the air to go through a humidifier. The two flows joined and passed through a 2.3 L glass mixing chamber, and then proceeded towards the filtration unit at 3 Lpm, corresponding to a fac e velocity of 5.3 cm/s, which is a standard value use d in military filtration testing (US Army, 1999). Based on this velocity, the residence time through the mixing chamber was 0.21 s, which was sufficient for microbes to come into equilibrium with the rel ative humidity. One of the two units was operated without a filter as a control and the other was equipped with the experimental variable, a circular filter with a diameter of 35 mm. The pressure drop across the filter, was measured using a Magnehelic gauge connected between upstream and downstream of the test filter The bacterial aerosols that penetrated the filter were collected in BioSamplers (SKC Corp.) containing 15 mL of 1X PBS at 5.5 Lpm for 5 mins and then 1m L of sample was assayed after serial dilution to get the countable range between 30 300 CFU/mL as mentioned before. E xperiments were conducted at RT (23 2 C) and two relative humidities (RHs): low RH (30 5%, LRH) and high RH (90 5%, HRH). For the virus, t he same conditions

PAGE 50

50 were employed, except for changes to DI water in lieu of 1X PBS as the collection medium and PFU instead of CFU. Physical removal efficiency was obtained by using the Aerodynamic Particle Sizer (APS; Model 3321, TSI Inc.) and Scanning Mo bility Particle Sizer (SMPS; Model 3936, TSI Inc.) for E. coli and MS2 ae rosol, respectively ( in Fig ure s 3 2 B and 3 2C ). Removal E fficiency, R elative S urvival F raction and St atistical A nalysis Removal efficiency of the test filters for water filtration c an be expressed as viable removal efficiency. For air filtration, both physical removal efficiency and viable removal efficiency were considered. Removal efficiency was calculated according to Eq uation 3 1: Removal efficiency (%) = ( 3 1) where N C is the concentration for the control part and N E is the concentration for the experiment part. The viable removal efficiency was obtained by comparing the microbial concentrations of original suspension ( Nc ) and filtered one ( N E ) for water f iltration and the concentrations of control ( Nc ) and experimental BioSampler ( N E ) for air filtration. For physical removal efficiency Nc was the number of particles entering the test filter and N E was the number of particles exiting the test filter obtain ed by APS or SMPS. The filter quality factor, a useful criterion for comparing filters, based on the physical removal efficiency of the test filters at 5.3 cm/s face velocity was calculated according to Eq uation 3 2 Quality factor (3 2) where p is the particle penetration. After the removal efficiency experiments, the test filters were taken off from the filter holder in the experimental system and subjected to a wrist action shaker (Model 75, Burrell Scientific) to investigate the viability of microbes collected on the filter. The filter was placed in

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51 25 mL of autoclaved DI water in a 50 mL conical tube and shaken for 15 mins. The survival fraction (SF), defined as the ratio of the microbe in the extraction solution to the total mi crobe count collected on the filter, was used to compare the results of the treated DAC filter with the untreated filter. Herein, the total microbes collected on the filter for air filtration tests were determined by the BioSampler count of the control sub tracted by the BioSample count of the experimental flow. Because the collection efficiency of Biosampler is dependent on air flow rate and particle size, relative survival fraction (RS) was introduced by comparing survival fractions of both filters to remo ve the effects of air flow rate and particle size: Relative survival fraction (RS) = (3 3) test on a completely randomized design with three or more replications with Design Expert 8.0 software. Results Water F iltration The experimental results, including viable removal efficiency and relative survival fraction, are summarized as averages of 5 trials and 3 trials, respectively, for each test fil ter in Table 3 1. For pure cultures (e.g., DI water and 1X PBS), there was no significant difference in viable removal efficiency among the four tests for E. coli although higher removal efficiency was expected because of increase of hydrophobicity after p eriodate reaction (Varavinit et al., 2001) However, the increased treatment time resulted in significantly lower relative survival fraction ( p =0.03) for E. coli For MS2, viable removal efficiency increased and relative survival fraction decreased signifi cantly ( p =0.005 and p =0.01) as treatment time increased. Compared with E. coli the slightly lower viable removal efficiency and higher relative survival fraction indicate the

PAGE 52

52 higher resistance of MS2 compared to E. coli For real water samples (i.e., the Suwannee river), real conditions such as emergency response or drinking water purification. In general, infectious bacteria and virus concentrations on the fi lter decreased as treatment time for the DAC filter increased, indicating the inactivation effect of the DAC filter through direct contact. Air F iltration The particle size distribution of E. coli and MS2 aerosols upstream of the test filters had a mode a t approximately 0.8 m and 28 nm respectively at LRH/RT as shown in Fig ure 3 3. The pressure drops across the untreated filter and three DAC filters (4 8 and 12 hr treated filters) are listed in Table 3 2. Since the pressure drop of the test filter at 3 Lpm was out of the measurable range of the Magnehelic gauge, the expected pressure drop of the filter was calculated by using the pressure drag ( S drag as shown in E quation 3 4 after measuring the pressure d rop of the filter at 1 Lpm. (3 4) where V f is the face velocity. As shown in Table 3 2, the pressure drop of the treated filter was lower than that of the untreated filter. The effect of RH on the pressure drop was negligible (dat a not shown). During the filtration experiment, variation in the pressure drop of the test filters was negligible. Regarding the physical removal efficiency, the 12 hr treated DAC filter presented the lowest value among the test filters as a function of pa rticle size, and the most penetrating particle size shifted to smaller size (~110 nm for untreated filter vs. 30 nm for 12 hr treated filter) as shown in Fig ure 3 4. The quality factor is also presented in Table 3 2. As shown, increasing the treatment time for the DAC filter increased the quality factor for the virus, but led to a decreased quality factor for the bacteria.

PAGE 53

53 The removal efficiencies, relative survival fractions, and quality factors based on viable removal efficiencies of the untreated filter and the 12 hr treated DAC filter under various environmental conditions are displayed in Table 3 3. For E. coli RH was not an important parameter for the untreated filter in that the differences of the viable removal efficiency ( p =0.17) relative surviva l fraction ( p =0.14) and quality factor ( p =0.18) results under two different RH conditions were within experimental error. On the other hand, at HRH, the 12 hr treated DAC filter presented increased viable removal efficiencies ( p =0.002) compared to the res ults at LRH ( p =0.08) The possible reason is that moisture in HRH might react with conjugated aldehyde in treated DAC filter to make carboxylic acid which can react with amine structure of RNA (Song, 2008) The relative survival fraction of the 12 hr treat ed filter ( p <0.001) was significantly lower than that of the untreated filter at HRH whereas the difference was negligible at LRH ( p =0.12), indicating the antimicrobial effect of the DAC filter under moist conditions on the E. coli collected on the treated filter. Inhibiting survival and growth of microorganisms collected on the filter is critical in preventing reaerosolization of collected microbes from the filter. For MS2, increasing RH resulted in an increased viable removal efficiency for both the untre ated and the 12 hr treated DAC filter ( p =0.004 and p <0.001). Discussion Water F iltration Difference in the viable removal efficiencies of E. coli and MS2 was expected because their singlet sizes are very different. Contrary to that expectation, the differ ence was not significant even though E. coli has a much larger singlet diameter than MS2. One possible reason for this phenomenon is the increased size by aggregation and/or encasement of the virus in the feed solution. The aggregation made the virus behav e more like a larger particle, which in turn narrowed the difference between the bacteria and virus test results. The aggregation was

PAGE 54

54 confirmed by SEM and DLS, as shown in Fig ure 3 5. In studies related to aggregation of viruses in water, Floyd & Sharp (19 77) found that poliovirus, having a similar physical property to MS2, existed in the aggregation state in DI water. Riemenschneider et al. (2010) also showed MS2 aggregation in DI water at high concentrations. As displayed previously, the pressure drop of the filter decreased as treatment time increased, which indicates increases in porosity and also possible changes in mechanical filtration efficiencies. Extraction efficiency was originally expected to be small due to the high retention capability of the resin filter reported in Lee et al. (2009) In contrast, the SEM images (Fig ure 3 6) of E. coli loaded on the 12 hr treated DAC filter before and after agitation treatment show that most microbes collected on the filter were extracted successfully after wri st action shaking The possible reason for the difference is dissimilar filter materials (cellulose filter vs. quaternary ammonium resin). Air F iltration The shift of the most penetrating particle size along with lower pressure drop of the treated filter than that of an untreated filter suggests possible morphology change of the filter media by treatment. This suggestion is supported by the FT IR spectra of 12 hr treated DAC filter using untreated cellulosed filter as background as shown in Fig ure 3 7. Thr ee characteristic bands of DAC appeared around 1730, 960, and 880 cm 1 The sharp peak at 1730 cm 1 is a characteristic band of carbonyl groups and the broad bands at 960 and 880 cm 1 are assigned to the hemiacetal and hydrated form of the DAC filter, indi cating increasing oxidation levels. In addition, a weak absorbance at around 1690 cm 1 corresponds to the conjugate aldehyde stretching, representing degradation of cellulose (Kim, & Kuga, 2004) In general, these increased bands represented increased oxid ation level. The oxidation degree by comparison of the absorbance of two bands, C=O stretching and CH 2 stretching, was 50% for the 12 hr treated filter (Bruck, 1991) Figure 3

PAGE 55

55 8 displays the SEM images of the filters before and after treatment. As seen, in creased pore size was apparently confirmed. Although the removal efficiency of the treated filter was lower due to increased pore size distribution, the pressure drop was also lower. For fair comparison, the viable removal efficiency was calculated for a t hicker 12 hr treated filter with the same pressure drop as the untreated filter using the quality factor relationship (Eq uation 3 2). The corresponding viable removal efficiencies of 12 hr treated filter at LRH for E. coli and MS2 are 97.56 % and 94.77 %, respectively. As shown, they outperform the untreated filter even at LRH (Table 3 3). The difference between physical removal efficiency and viable removal efficiency of the untreated filters might be attributed to the different principles of measurement s and intrinsic properties of the microorganisms. APS and SMPS were applied to count particles physically for physical removal efficiency, whereas the single layer method was used to determine the concentration of only viable bacteria and viruses in the en tire range for viable removal efficiency. Aggregated viruses counted as a single particle by the SMPS can be assayed as several single viruses after dispersion in the collection medium, and empty droplet counted by SMPS does not register any count by the b ioassay. Since environmental conditions ( e.g. RH) affect the virus viability, the concentration of viable agents for viable removal efficiency also changes as RH varies. All these contribute to a difference between viable removal efficiency and physical re moval efficiency. As mentioned, the relative survival fraction is an important parameter in evaluating the reliability of the filter as an antimicrobial filter for air filtration and determining the mechanism. The relative survival fraction was used to fai rly compare the effect of RHs on inactivation. Accordingly, 1.03 and 0.43 for E. coli and 0.96 and 0.38 for MS2 were obtained at LRH and

PAGE 56

56 HRH, respectively. The fact that these ratios decrease as relative humidity increased for both microorganisms ( p <0.001 for E. coli and p =0.001 for MS2) suggests sufficient water contents are important to the biocidal effect of DAC. The relative survival fractions of other biocidal filters are listed in Table 3 4. Rengasamy et al. (2010) reported that four different bioci dal filters (i.e., silver copper, envizO 3 iodine, and TiO 2 treated filters) showed 0.25 0.79 and 0.001 0.08 of relative survival fractions at LRH and HRH, respectively. However, it should be cautioned to make any direct comparison with the DAC filter beca use of different incubation times and conditions. Lee et al. (2009) u sed similar test conditions to the current study for an iodine treated filter and reported a relative survival fraction of 0.63. The iodine treated filter exhibited insignificant differen ce among different RH conditions unlike the DAC treated filter. Another category of inactivation technology is the energetic method. Heimbuch et al. (2010) reported the relative survival fractions by energetic methods of microwave generated steam, UV, and moist heat to be less than 10 5 for H1N1 virus. Although the inactivation performance of the DAC treated filter was lower than those energetic methods, it requires no additional energy source as mentioned earlier. Summary The retail cost of 50 cellulose filters is around $60. The only chemical required for the filter treatment is sodium periodate, which costs less than $2 for treating 50 filters through the soaking method. This 3% additional treatment cost to the original media yields a product with a hig her removal efficiency, lower pressure drop, and better disinfection capability. These are all important attributes of an effective biological filtration medium for practical applications such as respiratory protection, ventilation, and water purification system.

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57 Table 3 1 Viable removal efficiency and relative survival fraction of untreated filter and three DAC filters treated under different treatment times in water filtration. Test medium Test filter Test microbe Viable rem oval efficiency (%) Relative survival fraction 1X PBS Untreated E. coli 89.92 6.14 1.00 0.24 4 hr treated 88.24 5.72 0.44 0.05 8 hr treated 90.51 6.68 0.29 0.02 12 hr treated 92.34 5.04 0.15 0.01 Suwannee River Untreated E. coli 91.55 1.04 1.00 0.24 12 hr treated 92.87 1.27 0.15 0.10 DI water Untreated MS2 81.27 2.03 1.00 0.07 4 hr treated 83.64 3.36 0.63 0.19 8 hr treated 86.90 2.00 0.32 0.07 12 hr treated 88.63 1.12 0.28 0.02 Suwannee River Untreated MS2 84. 46 1.23 1.00 0.07 12 hr treated 88.95 1.27 0.28 0.18 Average for *5 trials and 3 trials Table 3 2 Pressure drop and quality factor based on physical removal efficiency for four filters at HRH Test filter Press ure drop at 5.3 cm/s (Pa) Quality factor (kPa 1 ) MS2 E. coli Untreated 3936 0.54 3.5 4 hr treated 3116 0.68 NM 8 hr treated 2952 0.72 NM 12 hr treated 1619 0.98 1.56 Not Measured

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58 Table 3 3 Removal efficiency, relative survival fraction, and quality factor based on viable removal efficiency of untreated filter and 1 2 hr treated DAC filter at two relative humidities in air filtration system Test microbe Relative humidity Test filter Physical removal efficiency (%) Viable removal efficiency (%) Relative survival fraction Quality factor *, # (kPa 1 ) E. coli LRH Untreated 99.9999 0.0001 82.1 2.6 1.00 0.33 0.44 0.04 12 hr treated 92.0663 2.4392 78.3 4.9 1.03 0.34 0.94 0.16 HRH Untreated 99.9999 0.0000 79.8 1.7 1.00 0.42 0.41 0.02 12 hr treated 99.7137 0.1172 89.2 3.7 0.43 0.05 1.37 0.26 MS2 LRH Untreated 92.3401 72.1 5.3 1.00 0.06 0.32 0.05 12 hr treated 78.5355 70.3 2.4 0.96 0.10 0.78 0.02 HRH Untreated 83.1872 88.1 3.3 1.00 0.02 0.54 0 .08 12 hr treated 80.8232 93.3 6.7 0.38 0.12 1.59 0.40

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59 Table 3 4 Comparison of other disinfection technology Target microbe Disinfection technology Condition Relative survival fraction MS2 Biocidal filter Silver copper En vizO 3 sheied Iodinated resin TiO 2 30 min loading/ 22 o C/ 30% RH/ 2 hrs 0.63 0.79 0.63 0.25 Silver copper EnvizO 3 sheied Iodinated resin 30 min loading/ 37 o C/ 80% RH/ 4 hrs 0.04 0.08 0.001 MS2 Biocidal filter Iodinated resin 30 mins/ In flight test/HRH 0.63 H1N1 # Energetic method Microwave generated steam 1250 W/ 2 mins 1.310 5 Ultra violet germicidal 1.6 2.0 mW/cm 2 / 15 mins 5.010 6 Warm moist heat 65 o C/ 85% RH 1.010 5 Rengasamy et al. 2010 Lee et al. 2009 # Heimbuch et al. 2010

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60 Figure 3 1 Experimental set up for the removal efficiency of the test filter in water filtration

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61 Figure 3 2 Experimental set up: A) viable removal efficiency and physi c al removal efficiency against B) E. coli and C) MS2 of the test filter in air filtration

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62 Figure 3 3 The number based particle size distribution of aerosols entering the filter at room temperature and low relative humidity Figure 3 4 Physical removal efficiency of four different filters as a function of particle size at room temperature and low relative humidity

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63 Figure 3 5 Particle size distributio n of the MS2 virus titer of 10 9 PFU/mL by A) SEM image and B) particle size distribution by DLS

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64 Figure 3 6 SEM images of the 12 hr treated DAC filter with collected E. coli before and after extraction

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65 Figure 3 7 FT IR spectra of 12 hr treated DAC filter when untreated filter was used as background

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66 Figure 3 8 SEM images: A) untreated DAC filter and B) 12 hrs treated DAC filter

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67 CHAPTER 4 US E OF DIALDEHYDE STAR CH TREATED FILTERS F OR PROTECTION AGAISN T AIRBORNE VIRUSES Background The increasing threat of pathogenic virus outbreaks such as Severe Acute Respiratory Syndrome (SARS), avian flu, and the more recent swine flu have spurred the publ in regards to the health related issues and protection methods against viral aerosols (CDRF, 2006). Filters are one of the most commonly used devices to remove viral aerosols because of their affordability, ease of application, and effectiven ess ( Fisher et al., 2009) Filtration efficiency is determined by several mechanisms such as interception, impaction, diffusion, gravity, and electrostatic attraction depending on fiber density, diameter, filter thickness, and other factors (Hinds 1999). However, these mechanisms are only related to the physical capture of viruses onto the substrate. Conventional filters s till allow these collected viruses to infect others if r eaerosolized through the respiratory processes of exhalation, sneezing, or coug hing, or as a fomite (CDRF, 2006). Filters can be modified by i ncorporating chemicals as antimicrobial agents to inactivate viruses (Gabbay et al., 2006; Silve r et al. 2006; Lee et al., 2009 ) A ldehyde s such as formaldehyde and glutaraldehyde are also we ll known antimicrobial agents, as they are highly reactive molecules which are able to combine with proteins and nucleic acids of microbes by cross linking and alkylation Nevertheless, their use as incorporated agents has been limited in the air filtratio n field because of the toxic effects if small aldehyde compounds were to be released and inhaled (Mcdonnell & Russell, 1999) For this reason, polymeric dialdehydes, such as dialdehyde starch (DAS), are promising alternatives. They not only react with hydr oxyl, Reprinted with permission from Woo, M H., Grippin, A., Wu, C. Y.& Baney, R. (2011) Use of dialdehyde starch treated filters for protection against airborne viruses J ournal of Aero sol Sc ence 46, 77 82

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68 amino, imino, and sulfurhydryl functional groups, resulting in the inactivation of viruses, but they also show very low toxicity (Mcdonnell & Russell, 1999; Radley, 1976). Our preliminary study demonstrated that a DAS aqueous suspension acted as an e ffective biocide against MS2 bacteriophage, PRD1 bacteriophage, and poliovirus. Thus, it is of great interest to employ DAS to modify filters for effective and affordable protection against viral aerosols. The objective of this study was to explore the in activation efficiency and filtration performance of commercial filters modified by different DAS suspension concentrations. Three different types of filters, namely, two cellulose filters (CFs) that are commonly used for air cleaning and a polypropylene fi ltering facepiece respirator (PF), were modified with DAS aqueous suspension. Porosity of CFs was factored into the filter selection due to their influence on collection efficiency of viral aerosols. Material s and Methods Test F ilters DAS aqueous suspensi on was prepared by mixing DAS (Granular form, Sigma Aldrich, P9265) with deionized (DI) water and then cooked at 95 o C for 2 hrs, as described in Song et al. (2009). After heat treatment to allow the granules to release the DAS poly meric molecules (Veelaer t et al. 1997), gel formation was observed. Coarse pore cellulose filters (CCFs, Whatman No. 54 with a thickness of 185 m and a pore size of 22 m ) fine pore cellulose filters (FCFs, Whatman No. 50 with a thickness of 115 m and a pore size of 2.7 m ), and facepiece respirators made of polypropylene (PFs, Dupont 01 361 N) were employed in this study. Test filters were pre pared by immersing filters into 100 mL of the suspension with different concentrations ( i.e., 1%, 2%, 3%, and 4% ) for 5 mins. These filters were immersed in the same volume of DI water overnight and then washed several times

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69 with DI water to rinse out exce ss DAS. After removing the residuals, the filters were dried at room temperature overnight. In this study, 4% was selected as the maximum concentration because in preliminary test, DAS treated filters with higher concentrations were fragile, indicating uns uitability for air filtration. Test A gent MS2 bacteriophag e (MS2; ATCC15992 B1 TM ) which is a commonly used surrogate for human pathoge n enterovirus (e.g., rotavirus) due to their similarities in resistance to antimicrobial agents, was used as the test age nt MS2 is easy to prepare and assay and it only requires a bio safety level 1 facility. Freeze dried MS2 was suspended in sterile DI water and diluted to a titer of 10 8 10 9 plaque forming unit (PFU)/mL. Artificial saliva was used as the nebulized medium to emulate aerosols produced from sneezing or coughing Details of the recipe for artificial saliva are reported in Woo et al. (2010) Experimental M ethod A schematic diagram of the experimental set up for aerosol filtration is displayed in Fig ure 4 1 M S2 aerosol was generated by a six jet Collison nebulizer (Model CN25, BGI Inc.) that had a virus titer of 10 8 10 9 PFU /mL at a flow rate of 7 Lpm with a pressure of 6 psi. The flow rate through the system was controlled by a rotameter, which was calibrated with a primary flow meter ( DryCal DC Lite, Bio International Co. ). The flow containing MS2 aerosol joined the other flow rate of 7 Lpm, passed through a humidifier for H RH con dition and then proceeded towards a 2.3 L glass mixing chamber Thereafter, the flow was split and each flow then reached the corresponding filtration unit at a flow rate of 3 Lpm This flow rate correspond s to the face velocity of 14.2 c m/s, which is a standard value for respirator filtration testing ( NIOSH, 1995 ). The excess air pa ssed through a separate line and bypassed the test filter. The viral aerosols that penetrated 22 mm ) were collected by BioSamplers (SKC Corp.) containing 15

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70 mL of sterile DI water at a total flow rate of 5.5 Lpm for 30 mins for the exp erimental line. The reason for using a lower flow rate than the standard 12.5 Lpm is to avoid significant reaserosolization of MS2 at high flow rate ( Riemenschneider et al 2009) An empty filter holder without a filter was used for the control line. The c ollected sample was assayed with E.coli as a host by the single layer method (EPA, 1984). The viable removal efficiency (VRE) was determined by comparing the PFUs from the experimental and control BioSamplers: VRE = (4 1) where N C and N E are the viral concentration s collected by the control and the experimental BioSamplers, respectively To quantify the amount of MS2 virus collected in a given filter, the test filter taken off from the system was immersed into 25 mL of sterile DI wa ter as an eluent and shaken by a wrist action shaker (Model 75, Burrell Scientific) for 15 mins. To evaluate the biocidal efficacy of treated filters compared to untreated filters, relative survivability (RS) of viruses on filters was calculated by compari ng survival factors, SFs, of both filters as: RS = (4 2) Herein, SF is defined as: SF = (4 3) where N S is the viral concentration obtained f rom the eluent, EE is the extraction efficiency of microbes from f CE is the collection efficiency of the Biosampler and V E and V B are the liquid volumes of the eluent and the Biosampler, respectively Consideration of the extraction efficiency and the collection efficiency of the BioSampler was not necessary fo r this relative

PAGE 71

71 value, RS. The pressure drop across the filter was measured by a Magnehelic gauge to evaluate filter performance using quality factor (QF) a useful criterion for comparing filter performance of different filter type s and thickness es This value is defined as (Hinds, 1999): QF (4 4) where P is the pressure drop It should be noted that throughout the duration of experiment (30 mins), the variation of the pressure drop was negligible. Experiments were carried out in t riplicate and samples were assayed in duplicate. S tatistical analysis was conducted using 2 way analysis of variance ( 2 way ANOVA) with Design Expert 8.0 software. Additionally, a s canning electron microscope (SEM, JEOL JSM 6330F, JEOL Inc.) was used to o bserve morphological changes of the filters. Results and Discussion CCFs and FCFs slightly shrank after the DAS treatment, whereas no visible change was observed for PFs. Also, when CFs were subjected to the wrist action shaker, untreated filters were brok en after 15 mins whereas treated filters remained intact. This is presumably because of crosslinking or entanglement of DAS with the cellulose. DAS can react with cellulose of CCF and FCF by forming covalent hemiacetal and acetal bond (BeMiller and Whistle r, 2009). SEM images of untreated and treated filters with different DAS suspensions, as shown in Fig ure 4 2, confirm this hypothesis. CFs are composed of main fibers and fibrillates of small diameter. CCF contains small amounts of fibrillates, whereas FCF has numerous fibrillates. DAS was crosslinked with fibrillates of CFs, resulting in the increase of fibrillate diameter (Fig ures 4 2D 4 2I) G ranular DAS w as also observed on the surface of the main fiber s at high DAS concentration (Fig ures 4 2D and 4 2H 4 2 I ) In contrast, DAS could not react with fibers of PF due to the lack of reaction between hydrophobic propylene filters and hydrophilic DAS (Song,

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72 2008 ; Yu et al., 2010 ) Instead, the DAS with gel formation might coat the fiber surface of PF res ulting in slight increase in fiber diameter. Also, the hy drophilic property by DAS coating might be the possible reason of the folded structure as shown in Figure 4 3C (Yu et al., 2010) The difference in materials yields differences in the impact of the D AS treatment, as will be demonstrated in the following section. The pressure drop results are listed in Table 1. Both filter type ( p < 0.0001) and DAS concentration ( p < 0.0001) are significant as well as the interaction between filter type and DAS concent ration. Since the pressure drop of FCFs at 3 Lpm w as out of the measurable range, the pressure drop at 0.5 Lpm was measured and then converted for 3 Lpm by Equ ation 4 5 2 = (4 5) where V is face velocity The pressure drops of unt reated and DAS treated PFs were less than 1 inch H 2 O at a face velocity of 14.2 cm/s. These values were much lower than the inhalation resistance of 2.52 inch H 2 O permitted by NIOSH for certified respirators (NIOSH, 1995) and the military standard of 4.21 inch H 2 O for HEPA filter media (U.S. Army, 1998). Furthermore, there was no significant change in pressure drop as a function of DAS treatment. The pressure drop of untreated CCF was 3.45 inch H 2 O, which is not suitable for the application of a respirator. However, those of treated CCFs ( p < 0.0 001 ) significantly decreased with increases of DAS concentration. Finally, those treated with the 4% DAS suspension reached the NIOSH limit for respirators. Meanwhile, the pressure drop of untreated FCF was too high to use as a ventilation filter and respirator, but those of the treated FCFs ( p < 0.00 0 1) significantly decreased although the pressure drops were still over the criterion for respirators. F igures 4 3A and 4 3B show that the VRE and QF of the test filters as a function of concentration of DAS suspension. For the PF, the DAS treatment did not change the fiber

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73 diameter of the filters, resulting in insignificant change in VRE. Hence, there was no significant change in QF. For the CF, the treatment did not cau se discernible impact on the VRE although the treatment reduced air resistance as exhibited as decrease d pressure drop. Accordingly an increase in the QF was observed. The QF s of untreated and 4% DAS treated CCFs were 0.77 and 1.46 kpa 1 and those of FCFs were 0.25 and 1.85 kpa 1 respectively. The impact on FCF (e.g., increase of 7.4 times) was larger than on CCF (e.g., increase of 1.9 times). The amount of crosslinking of the fibrillates is likely responsible for the difference. To evaluate the inactivat ion capability of DAS treated filter mats, the RS of treated filters as a function of concentrations of DAS suspension is displayed in Fig ure 4 4. All filters show similar tendencies, with RS decreasing with increasing concentration of DAS suspension, indi cating the inactivation effect of the DA S treated filter s against the MS2 collected on the treated filter. A ldehyde functionality of polymeric molecules dispersed from gel formation through heating in preparation is the source attributed to the biocidal ca pacity on treated surfaces. (Song, Cruz, Farrah, & Baney, 2009) Summary T his study demonstrates that treating filters with inexpensive DAS (less than $1/ 10 filters) is a practicable method for creating biocidal filters. DAS treatments have different impac ts on filtration efficiency depending on filter materials. The treatment reduces air resistance in cellulose filters, resulting in lower pressure drop while having no significant impact on removal efficiency. These factors combine to yield an improved qual ity factor for cellulose filter. Meanwhile, there is no difference in quality factor for treated propylene filters. Nevertheless, the biocidal effect is clearly exhibited for all treated filters. Compared to other decontamination methods, the DAS treatment does not require additional energy sources and facilities (e.g., microwave, IR, and UV irradiation) and it does not release toxic chemicals (e.g., iodine treated

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74 filter). Therefore, the treated filters can be adopted for wide applications such as in healt h care facilities and at pandemic events.

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75 Table 4 1 Pressure drop (face velocity of 14.2 cm/s) of three types of filters treated with different concentrations of DAS suspension. Concentration of DAS suspension Pressure Drop (i nch H 2 O) PF CCF FCF 0% 0.69 0.01 3.45 0.00 32.40 0.76 1% 0.69 0.01 3.25 0.07 27.00 1.27 2% 0.64 0.01 3.15 0.00 25.92 0.01 3% 0.65 0.00 2.95 0.07 8.10 0.21 4% 0.63 0.04 2.53 0.04 3.24 0.00 *Converted from 0.5 Lpm

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76 Fi gure 4 1 Schematic diagram of the experimental set u p : A) SEM image and B) particle size distribution by DLS of the MS2 virus titer of 10 9 PFU/mL

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77 Figure 4 2 SEM images: untreated A) PF, D) CCF, and G) FCF, treated B) PF, E) CCF, and H) FCF with 2% DAS suspension, and treated C) PF, F) CCF, and I) FCF with 4% DAS suspension. Magnifications of A) C) 500 and D) I) 1000 respectively

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78 Figure 4 3 Performance of filters treated with different concentrations of DAS suspension: A) viable removal efficie ncy and B) quality factor

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79 Fi gure 4 4 Relative survivability of MS2 viruses on filters treated with different concentrations of DAS suspension

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80 CHAPTER 5 MICROWAVE IRRADIATIO N ASSISTED HVAC FILT RATION FOR INACTIVAT ION OF VIRAL AEROSOLS § Background Microw aves electromagnetic waves with frequencies between 300 MHz and 300 GHz are widely applied in food processing, wood drying, plastic and rubber treating, curing and preheating ceramics as well as in cleanup processes (Park et al., 2006). Microwaves are non ionizing but sufficient to cause the molecules in matter to vibrate, thereby causing friction, which is subsequently transformed into heat for various applications. Among the diverse applications, the use of microwaves for decontamination was studied soon after microwaves became available. Goldblith and Wang (1967) and Fujikawa et al. (1992) compared the effect of microwave irradiation on Escherichia coli ( E. coli ) and Bacillus subtilis ( B. subtilis ). They concluded that the heat produced was a key factor f or inactivating the bacteria in solid and aqueous phases. Meanwhile, there has been research demonstrating additional effects, beyond the purely thermal mode of inactivation. For example, distortion of membrane structure and function (Phelan et al., 1994), altered enzyme activity (Dreyfuss and Chipley, 1980), disruption of weak bonds (Betti et al. 2004), increased release of various substances (Campanha et al., 2007; Celandroni et al., 2004; Woo et al., 2000), and increased ionic strength due to an increase d current within cells (Watanabe et al. 2000) have all been reported. However, all of the aforementioned research was conducted in the liquid, solid, or aqueous phase. In recent years, microwave inactivation of airborne microorganisms has gained more int erest because of increasing concerns about health related issues regarding outbreaks of pathogenic airborne viruses (e.g., SARS, H1N1 and swine flu). For examples, Hamid et al. § Reprinted with permission from Woo, M. H., Grippin, A., Wu, C. Y., & Wander, J. Microwave irradiation assisted HVAC filtration for i nactivation of viral aerosols Accepted to Aerosol & Air Quality Research

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81 (2001) measured 90% inactivation efficiency (IE) by applying microwave irradiat ion to heterogeneous airborne bacteria and fungi at 600 W for four periods of 2.5 min, each separated by 5 min from the next. Elhafi et al. (2004) demonstrated that infectious bronchitis virus, avian pneumovirus, Newcastle disease virus, and avian influenz a virus were inactivated on dried swabs in less than 20 s at 1250 W. Another study, Wu and Yao (2010 a ) reported IEs of 65% and 6% against airborne B. subtilis var niger spore and Pseudomonas fluorescens respectively, in an air stream after exposure to mic rowaves at 700 W for 2 min and Wu and Yao (2010 b ) showed gene mutation through polymerase chain reaction denaturing gradient gel electrophoresis after microwave application. Other recent studies (Heimbuch et al., 2010; Zhang et al., 2010) have focused on m icrowave inactivation of contaminated filters. Although filters are effective devices for capturing bioaerosols, and they are utilized in virtually all modern heating, ventilation and air conditioning (HVAC) systems to reduce the spread of infectious virus es and also as filtering facepiece respirators (FFRs) at healthcare facilities, they are limited as a preventive method because they inactivate neither viruses that pass through the filter nor those that are captured As some pathogens have a low i nfectiou s or lethal dose, viruses that penetrate or reaerosolize have the opportunity to infect people the filter was intended to protect (McCrumb, 1961). Heimbuch et al. (2010) reported that microwave generated steam at 1250 W for 2 min induced a 5 log IE for H1N 1 virus collected on FFRs. Zhang et al. (2010) demonstrated that microwave irradiation could provide an adequate method for inactivating E. coli and B. subtilis endospores via a microwave assisted nanofibrous air filtration system. The air quality of indo or has been determined by HVAC system because the pollutants including infectious viruses exhaled by human and transmitted by objects can be mitigated

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82 through the filtration and recirculation by HVAC system. If the viruses captured on the filter and transm itted during recirculation in HVAC system can be inactivated, the risk of spread of virus can be reduced However, no research has been conducted to evaluate the applicability of this technology to commercial HVAC filters even though these filters are comm only used in hospitals and residential buildings for collective protection. Therefore, the objective of this study was to evaluate the inactivation performance of microwave irradiation assistance to HVAC filtration systems during in flight filtration again st MS2 bacteriophage ( MS2 ) Key parameters examined were microwave power levels, microwave application times, and relative humidity. The thermal stability of the filter media was also investigated. Material s and Methods Test F ilters and A gent ** Two commerci al HVAC filters made of polyethylene and polypropylene (Filter 1; 3M) and synthetic polymer (Filter 2; True Blue) were selected as test filters and glass microfiber LydAir MG (Filter 3; Lydall) was used for comparison. MS2 (ATCC 15597 a test agent. It is a surrogate for enteroviruses such as rotavirus because of their similar structural properties and resistance to heat and chemicals (Brion et al., 1999; Prescott et al., 2006). Freeze dried MS2 was suspended in DI water with a titer of around 10 8 10 9 plaque forming units (PFU)/mL as the virus stock suspension. Experimental S ystem A microwave oven ( Panasonic, NN T945SF, 2.45 GHz, continuous irradiation) with two one inch holes in the backside was used in this study Because common filte r holders could not survive in the microwave, a custom made quartz filter holder was placed inside the microwave. ** Polyacrylonitrile (PAN) and cross linked PAN nanofiber filter were tested and the results are displayed in Appendix B.

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83 To support the filter material and to enhance heat transfer, a SiC disk was employed inside the quartz reactor The experimental set up for t esting the inactivation o f the virus is shown in Figure 5 1 Six Lpm of dry air was passed through a six jet Collison nebulizer (Model CN25, BGI Inc. MA ) to aerosolize the viruses A second air stream passed through the humidifier and then rejoined the fl ow. After the combined flow passed through the mixing chamber, it was split three equal ways and each stream proceeded toward the filtration unit at 4 Lpm, corresponding to a face velocity of 5.3 cm/s, which is a standard face velocity for ventilation sys tem testing (U.S. Army, 1998). Of the three flows, two were directed to filter holders outside the microwave, one with and one without an HVAC filter as controls. The third was equipped with a n HVAC filter 47 mm in diameter (effective diameter 40 mm for t he quartz reactor used) inside the microwave oven. The filters inside and outside the microwave oven were labeled A and B, respectively. The BioSamplers downstream of the microwave/filtration system and non irradiated filter were labeled C and D, respectiv ely. The BioSampler downstream of the empty filter holder (control) was labeled E. For in flight microwave decontamination, microwave irradiation was applied for three 10 min cycles that included selected periods of irradiation 1, 2.5, 5 and 10 min/10 min at three different microwave power levels 125, 250 and 375 W. To select the microwave application conditions, the thermal stability of three test filters was analyzed with TGA/SDTA (851E, Mettler Toledo Inc., OH), and the temperature of filters on the SiC disk under different applied conditions was measured with an IR pyrometer (OS533E, Omega Engineering Inc., CT). After irradiation, the test filter was taken off the filter holder in the experimental system and subjected to wrist action shaking (Model 75, Burrell Scientific, PA) with a shaking angle of 20 for 15 min

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84 to extract the viruses (Woo et al., 2010). The extracted MS2 was assayed with E. coli as a host by the single layer method (EPA, 1984). For enumeration of MS2 viruses within an adequate count r ange of 30 300 PFU/mL, 1 mL of diluted MS2 was mixed with 9 mL of #271 agar and 1 mL of #271 medium with log phage E. coli and poured into the Petri dish. The mixture was solidified and then the plate was stored in the incubator at 37 o C for one night befo re counting. T he effectiveness of this process was evaluated by using two parameters: survival fraction (SF) and IE. The SF under microwave irradiation was calculated by comparing the viable MS2 in the two filters: SF = (5 1) wher e C A and C B are the viral concentrations collected by filters A and B, respectively. Viral aerosols penetrating the test filters under microwave irradiation were collected in BioSamplers containing 15 mL of DI water. The IE through the microwave/filtration system was obtained by comparing the viable MS2 concentration in the two BioSamplers: IE = (5 2) where C C and C E are the concentrations of viruses collected in the BioSamplers C and E, respectively. The filtration efficiency of th e filter itself (1 C D / C E ) was used to confirm the stability of this system after each test. The pressure drop of the filter was measured by a Magnehelic gauge to evaluate the degradation or change of filters after decontamination test. Triplicate experimen ts and duplicate assays were carried out, and 1 way ANOVA was used for statistical analysis after confirming over 90% of normality (Design Expert 8.0)

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85 The scanning electron microscopy (SEM, JEOL JSM 6330F, JEOL Inc., MA) images of virus contaminated filt ers were taken after conventional oven heating and after microwave irradiation heating to investigate non thermal effects of microwave irradiation. A conventional oven (ISOTEMP oven 230G, Fisher Scientific, PA) was used to provide purely thermal effects. Filters contaminated with a virus suspension of 10 10 PFU/mL of DI water were either microwaved or inserted into a conventional oven for 30 mins. Results and Discussion Temperature M easurement of T est F ilters TGA was used to determine the appropriate tempe rature range for microwave assisted HVAC filtration because of the concern that the polymer fiber of the filter might experienc e melting or other mutation s during the thermal process. For filter s A and B two endothermic events were observed at 125 130 o C and ~170 o C while for filter C no endothermic or exothermic event was observed over the 25~300 o C range (results not shown) Therefore, the temperature of 125 o C was selected as the maximum temperature for microwave irradiation to avoid filter TGA /DTA was used to determine the appropriate temperature range for microwave assisted HVAC filtration because of the concern that the polymer fiber of the filter might experience melting or other mutations during the thermal process. As displayed in Fig ure 5 2A no residual moisture loss around 100 o C was observed in all three filters, confirming the hydrophobicity of filter material. For filters 1 and 2, two endothermic events were observed at 125 130 C and at ~170 C; for filter 3 no endothermic or exothermic eve nt was observed over the 25~300 C range as shown in Fig ure 5 2B. Therefore, 125 C was selected as the maximum temperature for microwave irradiation to avoid filter damage. The temperature profiles of the filters supported on a SiC disk running with a fl ow rate of 4 Lpm at different microwave power levels and application times are displayed in Fig ure 5 3 A linear increase in temperature as

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86 application time increased was expected. However, the results showed a different trend. At 250 W, the temperature did not increase much after 2.5 min, likely due to the balance between heating by microwave irradiation and cooling by the air stream. At 375 W for 10 min/cycle, the max temperature was around 120 C, whereas it reached 165 C without airflow, illustrating th e cooling effect by the air stream. Based on the temperature measurement, a maximum power level of 375 W was selected to investigate the IE and SF in this study. For further investigations, higher power levels of 500 W and 750 W were selected for filter 3 because of its high thermal stability as mentioned previously. Inactivation E fficiency and S urvival F raction For filter 1 t he IE of the microwave irradiation assisted filtration system and the SF on the filter surface as a function of microwave power at four different microwave application times are displayed in Figure s 5 4 A and 5 4 B, respectively. As shown, IE increased and SF decreased as microwave power was increased and as the application time was extended. For the IE, changes to both microwave power level ( p< 0.01) and application times ( p< 0.01) were significant. The IE is attributed to two factors: 1) physical capture by the filtration mechanism and 2) inactivation of viruses during flight. At the lowest setting 125 W for 1 min/cycle no additional di sinfection was observed beyond the inherent log removal efficiency of 0.53 (i.e., 71%) coming from the physical filtration efficiency (1 C D / C E ) of filter 1 (~73%). At a power level of 375 W, 3.0 and 3.5 logs of the viable MS2 were disinfected when microwav e irradiation was applied for 5 and 10 min/cycle, respectively. This suggests that an application time of 5 min/cycle is sufficient to disinfect MS2. Significant influences of both microwave irradiation time ( p = 0.02) and power level ( p = 0.03) upon SF we re seen. The trends of SF were similar to those of IE, although a much lower SF was expected at higher microwave power levels because of the longer exposure time of 30 mins for the SF as compared to the shorter flight time of less than 5 s for the IE.

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87 Howe ver, at 375 W applied for 5 and 10 mins/cycle, a higher value of log IE was observed than the absolute value of log SF. Physical capture is one possible reason for the higher log IE. However, the log IEs after deducting the inherent removal efficiency wer e still higher than the absolute values of the log SF (2.5 and 2.9 vs. 1.8 and 2.5). This may be explained by the high temperature of the SiC disk. Damit et al. (2010) reported that the exposure of MS2 to the high temperature of 250 C for 1 s resulted in 4 log SF. The temperatures of the SiC disk at 375 W immediately after irradiation at 5 and 10 min/cycle were 172 C and 203 C, respectively, whereas the temperatures of the filters on the SiC disk were 107 C and 117 C. Thickness of the SiC disk was 2.54 cm, and viruses flying through the disk were exposed to these high temperatures for 0.5 s. This exposure during flight could attribute to the higher IE. For filter 2, similar results were seen, as shown in Fig ures 5 4C and 5 4D although the inherent fil tration efficiency was slightly higher than that of filter 1. For filter 3, IE and SF at 375 W, 500 W, and 750 W are displayed in Fig ures 5 4E and 5 4F As shown, the IE and SF at 375 W are similar to those for filters 1 and 2, although the higher inherent filtration efficiency was around 95%. At 500 W and 750 W, log SFs of 3.47 and 4.23 were seen, respectively. The temperatures of filter 3 on the SiC disk at 500 W and 750 W for 10 min/ cycle were 143 C and 171 C, respectively. This result suggests that the thermal stability of filter material is an important factor for microwave disinfection application s Effective T emperature Comparing the microwave irradiation power level and application time data revealed that filter disinfection can be characterized by a threshold temperature, i.e., the temperature at which inactivation starts to increase sharply. Similarly an effective temperature, defined as the minimum temperature that must be reached for effective disinfection (2 log or greater), can also be iden tified. The threshold and effective temperatures can be estimated in Fig ure 5 5 which

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88 displays log IE and log SF as a function of the temperature reached after microwave irradiation application. The data pattern greatly resembles a two stage process an in itial accumulation of energy, and then a catastrophic release, simply indicating a threshold temperature has been reached. The IE remains unsatisfactory until the threshold temperature of around 90 C, and it reaches 2 logs at 109 C The SF also starts to rise around 90 C and reaches 2 logs at 116 C Once the filter reaches this temperature, effective disinfection of the virus can be assumed. IE and SF of each filter against MS2 can also be expressed as a function of temperature ( T ) via a log linear rela tionship above the threshold temperature, as displayed in Table 5 1. Although different intercepts and slopes were expected because of different inherent physical removal efficiency and thermal propert ies of filter, the difference was not significant ( p< 0. 05). Furthermore, when the intercepts of log IE were corrected by deducting the inherent filtration efficiency, the results showed no difference among all three filters ( p = 0.02). Therefore, in and near the temperature region studied, the IE and SF of MS2 for a microwave irradiation assisted filtration system can generally be expressed as: log (IE) log (IE) inherent filtration = log (IE) microwave = 7.57 + 0.08 T (5 3) log (SF) = 5.01 0.0 6 T (5 4) Thus, for an HVAC filter having 99.9% filtration effic iency to reach 6 log IE for MS2, the necessary temperature is ~ 132 C Although thermal effect was a major factor for microwave inactivation, Khalil and Villota (1989) compared the distortion of RNA subunits in Staphylococcus aureus after microwave and co nventional heat treatments, and found destruction of the 23S RNA by only microwave treatment, indicating the possibility of non thermal effect. In addition, Betti (2004) reported a non thermal effect of microwaves against plants and viruses at a sublethal temperature, and Wu

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89 and Yao (2010) confirmed visible changes of bacteria and fungi after microwave heat treatment by ESEM. Hence in this study non thermal effects were investigated by studying the morphological changes and SFs with and without microwaves at the same temperature. Fig ure 5 6 displays the temperature profiles of the conventional and microwave ovens. T emperatures of the conventional oven were selected based on the temperature profiles of the microwave oven at 250 W and 375 W. The conventional the microwave oven operated at 250 W, a steady state temperature profile was observed after 10 min. However, temperatures around 90 C at 250 W might be insufficient to inactivate MS2. Therefore, 3 75 W was selected for observations of morphological changes through SEM. Fig ure 5 7 displays SEM images of untreated, conventional oven treated, and microwave treated virus contaminated filters. As shown in Fig ures 5 7 B and 5 7C the heat of both the conv entional oven and the microwave oven made the water evaporate (or removed it in some other way), and then aggregation was observed. However, no significant difference in morphology was seen, even though the concentration of microwave treated viruses was lo wer than that of conventional oven treated viruses. SFs of viruses on the substrates after heat treatment by microwave oven and conventional oven were also compared but no significant difference was shown, indicating that no non thermal effect of microwave s can be elucidated in this study. Effect of R elative H umidities on I nactivation P erformance Relative humidity is another key parameter for the inactivation of viruses. However, when a SiC disk is used, it is difficult to determine the effect of relat ive humidity because of the overwhelming thermal effect of the SiC disk as compared to relative humidity. Relative humidity is another key parameter affecting the inactivation of viruses. However, when a SiC disk is used, it is difficult to determine the e ffect of relative humidity because of the overwhelming thermal

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90 effect of the SiC disk as compared to relative humidity. Therefore, to investigate the effects of relative humidity, a quartz frit was used as a support instead of a SiC disk. IE through the sy stem and SF on the filter surface as a function of microwave power level applied to filter 1 for 5 min/cycle under three relative humidities are displayed in Fig ures 5 8 A and 5 8B respectively. IE ( p = 0.01) significantly increased and SF (p<0.01) signifi cantly decreased as the application time increased. By design the quartz frit could not absorb microwave irradiation, which resulted in a lower filter temperature and less pronounced viral inactivation capacity compared to those with the SiC disk. Log IEs of 0.8, 0.9, and 1.3 were obtained at relative humidities of 30%, 60%, and 90%, respectively, at 500 W ( p <0.01) Unlike the results with a SiC disk, log IE corrected for filtration efficiency of the filter itself was lower than the absolute value of log S F, indicating that the lower temperature of the support is insufficient to inactivate MS2. At the higher power level, a high IE and lower SF were seen under high relative humidity, which may be explained by the mechanism of microwave irradiation. The highe r water content can contribute to more (Fisher et al., 2011) However, the relative humidity effect was not observed at 500 W for 10 min/cycle in Fig ures 5 8C and 5 8D The different phenomenon can be explained by the increased concentration of water vapor at higher temperature and the higher temperature itself. At high relative humidity, final temperatures were 27 C, 43 C, 66 C, and 81 C after 5 min/cycle, and 49 C, 62 C, 78 C, and 89 C afte r 10 min/cycle at 125, 250, 375, and 500 W, respectively. The results suggest that relative humidity is a significant parameter from 50~80 C and that it ceases to be significant above 90 C. Degradation of F ilters after M icrowave I rradiation Table 5 2 sh ows that p ressure drops of the test filters after microwave treatment were measured to inspect for degradation of the filter. Under the operating condition, the initial

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91 pressure drops (at 5.3 cm/s) of 0.45, 0.62, and 1.20 inches H 2 O for filters 1, 2 and 3, respectively, were maintained throughout several microwave irradiation tests at 375 W for 10 min/cycle. There was no significant difference in pressure drop between control and treated filters, indicating no melting or degradation. SEM images also showed no visible morphological changes. Comparison to O ther D isinfection T echnologies Numerous disinfection technologies, including energetic techniques and chemical treatments, with or without filtration systems have been studied. A study investigating bleach d isinfection with 0.1% sodium hypochlorite aerosol and UV germicidal irradiation (UVGI) with a wavelength of 254 nm achieved 2 log SF of MS2; however, bleach and UVGI have limitations of chemical release and low penetration, respectively (Vo et al., 2009). Rengasamy et al. (2010) and Woo et al. (2011) confirmed the inactivation effect of biocidal filters incorporated antimicrobial agents, e.g., silver copper, oxygen species, titanium oxide and dialdehyde, but these filters could not reached 2 log SF for 30 m ins. Compared to other filter disinfection technologies, the microwave assisted filtration system was effective without any filter damage and chemical formation. As direct disinfection technologies without filter, Kettleson et al. (2009) reported electros tatic precipitator (ESP) at 6 kV showed 2 log IE of MS2 and that at 10 kV could reach above 6 log IE. To compare these results with the present study is difficult because of different inactivation mechanisms (thermal effect vs. radical reacti on). However, ESP disinfection should be cautioned about the formation of ozone. Grinshpun et al. (2010) demonstrated dry heat treatments of MS2 at 125 C for 0.24 s resulted in 2 log IE. This value is similar to that corrected by deducting the inherent fi ltration efficiency was considered, confirming thermal effect of microwave.

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92 Summary T his study demonstrates that microwave assisted filtration is an efficient approach for inactivating viral aerosol s Microwave power and application time are key operating parameters for controlling the disinfection effectiveness of viral agents. Both factors combine to yield a threshold temperature of around 90 o C. Relative humidity is another pivotal parameter for viability of viruses at medium temperature, but it becomes insignificant at high temperatures above 90 o C. If thermally stable filter material is applied, a high inactivation efficiency of around 5 log through the system can be reached at lower temperatures compared to other dry heat treatments.

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93 Table 5 1 Linear relationship of the IE and SF of MS2 with temperature (T) Filter 1 Log IE = 7.14 ( 7.65) a + 0.087 T ( p =0.04) Log SF = 4.67 0.057 T ( p <0.01) Filter 2 Log IE = 6.69 ( 7.46) + 0.077 T ( p =0.02) Log SF = 4.81 0.060 T ( p <0.01 ) Filter 3 Log IE = 6.57 ( 7.60) + 0.078 T ( p <0.01) Log SF = 5.05 0.061 T ( p <0.01) All filters b Log IE = 6.83 ( 7.57) + 0.080 T ( p= 0.02) Log SF = 5.01 0.060 T ( p <0.01) a The values in parentheses in log IEs are the intercepts calculated with inhere nt filtration efficiency deducted b The relationships were obtained from all IEs and SFs above threshold temperature of three filters Table 5 2 Pressure drop (face velocity of 5.3 cm/s) of three filters after microwave treatmen t at 375 W for 10 mins/cycle Pressure drop (inH 2 O) No. treatment Filter 1 Filter 2 Filter 3 0 (Control) 0. 45 0. 62 1.2 1 0. 45 0. 62 1.2 2 0. 45 0. 60 1.2 5 0. 46 0. 60 1.2

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94 Figure 5 1 The experimental set up for microwave irradiation assisted fil tration.

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95 Figure 5 2 Thermal stability for three filters of A ) Thermogravimetric analysis (TGA) and B ) differential thermal analysis (DTA) curves for three filters.

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96 Figure 5 3 Temperature of the filters as a function of microwave application time a t three different microwave power levels

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97 Figure 5 4 Log inactivation efficiency and log survival fraction: A) and B) filter A, C) and D) filter B, and E) and F) filter C

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98 Figure 5 5 Microwave inactivation performance: A) Log inactivation efficiency and B) log survival fraction as a function of the temperature reached during microwave irradiation. Filter 1 for 125, 250 and 375 W and Filter 3 for 500 and 750 W

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99 Figure 5 6 Temperature of microwave and conventional ovens as a function of application time

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100 Figure 5 7 SEM images: A) untreated virus contaminated filter, B) conventional heat treated virus contaminated filter, and C) microwave treated virus contaminated filter. Magnifications of A) C) 50,000 (A) (B) (C)

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101 Figure 5 8 Log inactivation efficiency by microwave irradiation assisted filtration system and and Log survival fraction on filter surface as a function of microwave power level: A) and B) for 5 min/cycle and C) and D) for 10 min/cycle

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102 CHAPTER 6 EFFECTS OF RELATIVE HUMIDITIES AND AEROS OLIZED MEDIA ON UV DECONTAMINATION OF V IRAL AEROSOLS LOADED FILTER Background The increasing threat of bioterrorist attacks (e.g. ebola virus ) and recent outbreaks of airborne pathogenic infections (e.g. SARS, H 1N1, Avian Flu) have raised the level of public interest in biological aerosols and protection methods that prevent their spread ( Drazen, 2002 ; Prescott et al., 2006 ; CDC, 2009 ) F iltering facepiece respirators (FFRs) certified by the National Institute for Occupational Safety and Health (NIOSH) are mandated by 40 CFR 84 t o be worn by healthcare personnel and are recommended as a protective device for the general public during a pandemic event Several factors influence the effectiveness of the intended protection offered by the FFRs, including the bioaerosol transmission m ode and environmental conditions. Because aerosol size is a pivotal parameter for filtration efficiency understanding the transmission mode of viral aerosols is critical to protection of the public against major airborne pathogen pandemics. T hree critical transmission modes are recognized for the spread of infectious viruses ( CDRF, 2006 ) : (1) Droplet transmission, which results from infected individuals generating droplets containing microorganisms by coughing, sneezing, singing and talking. Droplets of various sizes produced by an infected person are propelled short distances through the air to a susceptible host, and the infection occurs through contact with a mucous membrane. ( 2 ) Aerosol transmission, which includes the dispersion of droplet nuclei that remain airborne in air after evaporation of droplet s and dust particles carrying the microorganism. Owing to the small size of the inhalable droplet nuclei and dust particles, disease can be widely spread by this mode. ( 3 ) Fomite transmission, which includes both Reprinted with permission from Woo, M. H., Anwar, D., Smith, T., Grippin, A., Wu, C. Y., & Wander, J. Investigating the effects of relative humidities and nebulized media on UV inactivation of viral aerosols loaded filter. Submitted to Applied & Environmental Engineering.

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103 direct body to body contact and indirect contact through a contaminated o bject (e.g., towel or mask ). Controlling this mode is a chronic problem in healthcare facilities. One infected patient serving as a source can easily infect other especially immun o compromised patients through activity in the facility. As implied above, the FFR is a device that efficiently captures viruses transmitted by droplet and aerosol modes. However, infectivity of the captured viruses persists which makes the contaminated FFR a fomite. In addition, reaerosolization of viruses captured on the FFR is a possibility; transient, high rates of air flow (e.g., coughing and sneezing) aggravate the probability of reaerosolization ( Rengasamy et al., 2010 ) NIOSH projec ted that during a 42 day influenza pandemic the healthcare sector alone would require over 90 million masks, a demand that could create a supply shortage that would leave millions in unnecessary danger of infection ( CDRF, 2006 ) One possible buffer against this threat, recovering and reusing FFRs after inactivating the viruses that the y capture and thus allowing them to be reused several times, has been shown to be technically feasible ( Heimbuch et al., 2011 ) and appears consistent with 29 CFR 1 910.134(h)(1) (OSHA, 2012). However, imprecision in the the Center s for Disease Control and Prevention (CDC) recommends continuous or intermittent wearing for no more than 8 hours and on ly until the mask becomes wet, dirty or contaminated, ceases to and the specification in 29 CFR 1910.134(d)(1)(ii) that respirators must be NIOSH certified (OSHA, 2012), a certifica tion that applies only to new disposable respirators, poses a procedural obstacle to implementation of this concept, even in a declared emergency (FDA, 2007), that remains to be resolved. Decontamination of used FFRs plays an important part in preventing b oth reaerosolization of viral particles collected on a filter surface and fomite transmission, and

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104 can extend filter life. Several inactivation methods, including microwave irradiation, UV irradiation and biocidal surfaces (Vo et al. 2009; Fisher et al. 2009; Lee et al., 2009; Zhang et al. 2010) have been shown to decontaminate viral aerosols collected on filters. A ntimicrobial agents such as phenols, alcohols, heavy metals, and quaternary ammonium compounds can be incorporated into air filters ; however, t he biocidal filters that have been tested release small quantities of toxic chemicals (Lee et al. 2009; Woo et al., 2011; Rengasam y et al. 2010 ), as can chloramines ( Gowda et al., 1981 ) The use of direct microwave irradiation to kill microorganisms t hrough thermal and non thermal effects has also been demonstrated in various studies in solid media but this method requires a microwave absorber (typically water, activated carbon or silicon ca r bide) and may damage the material ( Fisher et al. 2009 ; Heim buch et al., 201 1 ; Zhang et al ., 2010; Rengasamy et al. 2010 ) UV irradiation delivers sufficient energy to be a practical antimicrobial method. UV C irradiation is a recognized method for inactivating a wide variety of biological agents and in particula r airborne microorganisms ( Prescott et al. 2006 ). At a wavelength of 254 nm, a UV C photon striking a biological cell is selectively absorbed by one of adjacent pairs of thymine or uracil nucleotide bases in deoxyribonucleic acid (DNA) or r ibonucleic acid (RNA), causing them to form covalent bonds with each other and interrupting hydrogen bonds with adenine bases in the complementary DNA /RNA strain Pyrimidine dimers of thymine /uracil bases distort the shape of DNA /RNA altering the double helical structur e and preventing the accurately transcribing or replicating its genetic material, which ultimately leads to the death of the cell ( Perier et al. 1992 ; Kowalski, 200 9 ; Prescott et al. 2006 ). Many studies (Farhad, 2004; Memarzadeh et al., 2010; Ryan et al., 2010 ; Chuaybamroong et al., 2011 ) have reported that UV intensity, exposure time, lamp placement and air movement patterns influence its inactivation efficiency (IE) against microbes.

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105 A study by Viscusi et al (2009) investigating the effect of U V light on the performance of FFR s found no net change in the functionality of the masks after UV radiolysis. Most studies have focused on the relationship between decontamination efficacy and two parameters, UV intensity and exposure time ( Viscusi et al. 2009 ). None has considered other important parameters (e.g., relative humidity (RH) nebulized medium, and transmission mode) that influence susceptibility of viral agents collected on fibrous filters The objective of this study was to investigate the I E of UV irradiation against viruses collected through different transmission modes under various environmental conditions. For this, the filters were contaminated by two pathways (droplet and aerosol) using three spraying media (i.e., deionized (DI) water beef extract (BE) in filtered, sterile DI water and an artificial saliva (AS ) ) at three RH conditions ( i.e., low (LRH, 30 5%), medium (MRH, 60 5%) and high relative humidity (HRH, 90 5%) ) UV irradiation was applied a t a constant intensity of 1.0 mW/cm 2 for different time intervals Materials and M ethods MS2 P reparation MS2 bacteriophage (MS2; ATCC 15597 B1) was used as the test agent. MS2 27.5 nm ) has a non enveloped, icosahedral capsid ( Prescott, Harley, & Klein 2006 ; Brion & Silverstein, 1999) and is commonly used as a nonpathogenic surrogate for human pathogenic viruses (e.g., rotavirus, influenza, poliovirus, and rhinovirus ) for the following reasons : physical characteristic s similar to th ose of human pathoge nic viruses, need of only BSL 1 containment and ease of preparation and assay ( Brion & Silberstein 1999 ). A f reeze dried MS2 culture was suspended in DI water at a titer of approximately 10 1 1 10 1 2 plaque forming unit (PFU)/mL and this stock was stored at 4 C. The virus stock was successively diluted to 10 8 10 9 PFU/mL with 1X phosphate buffered saline (PBS) and used for the experiment.

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106 A single layer bioassay with a host of Escherichia coli (ATCC, 15597) was used to enumerate the infectious viruses (EPA, 1984) Tryptone yeast extract glucose broth (TSB 271) and culture medium 271 were prepared following the American Type Culture Collection (ATCC) procedure for MS2 assay. Freeze dried E. coli were suspended in 1X PBS, inoculated into a solidified agar plat e (1.5% agar) with a sterilized loop, and incubated at 37 C overnight. The single colony from the plate was transferred into TSB 271 and set to grow at 37 C overnight. Culture medi um 271 (100 mL) was inoculated with 0.3 mL of the E. coli culture from TSB 271 and incubated at 37 C for 3 hrs. A 1 mL aliquot of in oculated E. coli culture was added to a sterile conical tube containing 9 mL of soft agar (0.5% agar) in a water bath between 40 C and 50 C. One millimeter of MS2 was added to the tube containing E. coli and agar the mixture was shaken thoroughly, and then it was poured into a petri dish. To attain the countable range of 30 300 PFU/mL, serially diluted MS2 samples were used. After the agar hardened, the plate was inverted and placed in an incubat or at 37 C overnight. Plaques on each plate were enumerated and the titer of the sample was determined by multiplying the dilution factor times the plaque count Spraying M edium Three types of spraying media were tested, DI water, BE and AS. DI water was included to explore properties of the naked virus. BE (0.3 vol %) was used to provide nutrients, which can also contribute encasement Beef e xtract is a mixture of peptides amino acids, nucleotide fractions, organic acids, minerals and some vitamins deri ved from infusion of beef (Cote, 1999). AS (0.6 vol %) 0.3% m ucin from porcine stomach (Sigma Aldrich, M1778) plus salts in DI water as a mucus simulant was used to mimic human respiratory fluid. Mucin viscous glycoproteins comp rising approximately 75% ca rbohydrate and 25% amino acids li n ked via glycosidic bond s between N acetylgalactosamine and serine or

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107 threonine resides readily form s a gel in water (Bansil et al., 1995). The details of AS composition were reported in Woo et al. (2010). Droplet and A ero sol L oading S ystem The experimental set up for loading droplets and aerosols containing viruses onto the substrate is displayed in Fig ures 6 1 A and 6 1 B respectively. A 2.4 MHz ultrasonic nebulizer (241T, Sonear, Farmingdale, NY, USA) was used to generate droplets containing viruses with a flow rate of 2 Lpm. The MS2 suspension in the reservoir was prepared by dispersing 1 mL of stock solution in 25 mL of spraying medium (i.e., DI water, BE or AS). C ircular coupons 2.54 cm ) were cut from a 3M 1870 (NIO SH certified N95) FFR. The droplets produced entered the chamber and loaded onto the surface of FFR coupons for 5 min. Each filter was then cut into four equal quadrants for UV exposure. Droplet size is affected by environmental conditions such as RH and t emperature. L oading of droplets on to filter coupons was conducted at room temperature (20 3 C) and HRH. A Collison nebulizer (CN25, BGI Inc., Waltham, MA, USA) was used to generate the aerosol containing viruses, with a flow rate of 6 Lpm. The MS2 susp ension in the nebulizer was prepared by dispersing 2 mL of viral stock solution in 50 mL of nebulizer medium. The aerosol from the nebulizer entered the mixing chamber and was mixed with dry or wet air as appropriate to adjust RH. Loadings were applied at each of the three RHs (LRH, MRH, and deliver 4 Lpm, corresponding to a face velocity of 5.3 cm/s, a standard face velocity for air filter system testing (U.S. Army, 1998) After loading with aerosol for 30 min at the selected RH, the filter was removed and cut into equal quadrants to prepare for UV exposure. UV E xposure During UV exposure, the UV C (254 nm) lamp (UVG 11, Ultraviolet Products, Cambridge, UK) was adjusted t o a height of 10 cm. UV intensity of 1.0 mW/cm 2 was

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108 measured using a radiometer (PS 300, Apogee, Logan, UT, USA). The quadrants were placed on a petri dish in a chamber and exposed to UV for different exposure times (0 2 h) at the selected RH One quadrant used as a control was not exposed to UV; the other three were exposed to UV for different times. All were evaluated after the maximum exposure time for a fair comparison. D ry/wet air was fed into the chamber to adjust the relative humidity in the system After the maximum exposure time, each quadrant was placed in a 50 mL conical tube containing sterilized DI water and agitated with a wrist action shaker (Model 75, Burrell Scientific, PA) inclined 20 o for 15 min to extract MS2. The MS2 extracted was assay ed with the single layer method. IE was determined by comparing the count from the irradiated coupon with that from the paired control: (6 1) Triplicate tests for each condition and duplicate assay were conducted Two way analysis o f variance ( ANOVA ) and t hree factor ANOVA w ere used for statistical analysis (Design Expert 8.0). The coefficient of variation of amounts loaded on the quadrants was less than 20%. S canning electron microscopic (SEM, JEOL JSM 6330F, JEOL Inc.) images of f ilters contaminated with viruses generated in different media were taken and compared to investigate the protecti ve effect of solid components Results and Discussion Effect of T ransmission M ode with D ifferent M edia Log IE is plotted in Fig ure 6 2 as a fun ction of UV irradiation time for both droplet and aerosol modes with three different nebulizer media. HRH was applied for both loading and UV irradiation. For droplet transmission mode, the log IEs were 4.32, 2.32, and 1.98 after 60 min irradiation in DI w ater, AS and BE, respectively, whereas for aerosol transmission mode,

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109 the log IEs were 5. 01 2.68 and 2.32 in DI water, AS and BE, respectively. The IEs in this figure depend on three parameters: 1) UV irradiation time: Extending the irradiation time incre ased the IE because the UV dose increased. When the application time was changed from 30 to 60 min, the dose of UV irradiation doubled from 1.8 J/cm 2 to 3.6 J/cm 2 increasing the amount of damage to nucleic acids. 2) Transmission mode: IE for aerosol s was higher than for droplets. Water in the droplets absorbs UV (Prescott et al., 2006; Kowalski, 200 9 ), and shielding of viruses near the center of the aggregate likely also contributes to this trend. The size of droplets generated from the ultrasonic nebuliz er was around 9 et al. 2010) whereas aerosols from the Collison nebulizer measured 1 1 droplet at HRH is 0.0077 s at 20 C. As the residence time of aerosol in the mixing chamber was 0.21 s, these particles reached equilibrium during transit. However, the evaporation time of 9 C 0.63 0.7 s, is much longer than the residence time S o the larger droplets retain much of their water at contact. The equations used are as follow s (Hinds, 1999): (6 2) where R is the ideal gas law constant, p is the density of particle, d d is the droplet size, D v is the diffusion coefficient of water vapor molecule, M is the molecular weight of water T and p are th e temperature and pressure away from the droplet surface, i.e. the environmental conditions and T d and p d are the same conditions at the droplet surface. Room temperature (20 C) wa s applied for T and the equation below wa s used to determine T d (6 3)

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110 where S R is the saturation ratio. The partial pressure in kPa at a given temperature in K is calculated according to (6 4) 3) Spraying medium: IEs in AS and in BE were much lower than in DI water for both aero sol and droplet transmission. The likely reason for this difference is a protective effect caused by solids in both AS and BE. Based on the composition of the media, the volume fractions of solids in DI water BE and AS were 1 10 4 3.1 10 3 and 6 0 10 3 respectively after complete evaporation DI water has a much lower solid content. Th is mode of protection i s supported by SEM images, shown in Fig ure 6 3 of filter s contaminated with MS2 viruses aerosolized in different media. Images of MS2 generated in D I water and loaded on the filter ( Fig ure 6 3B ) show aggregates in the range 100 nm 1 m on the substrate. Riemenschneider et al. (2010) and Jung et al. (2009) reported MS2 aggregates of around 200 nm for the MS2 suspension and 30 200 nm for captur ed aeroso l particles MS2 aerosolized in BE instead of DI water was captured as oval to spherical features as shown in Fig ure 6 3C. As displayed in Fig ure 6 3D, precipitated BE solids formed a thick shell encapsulat ing the MS2 virions and/or aggregates. The solids in AS are water insoluble mucin and various water soluble salts. To test the hypothesi s that the salts and mucin act separately to afford protection (Lee et al., 2011), MS2 was aerosolized from AS media prepared both with and without mucin. As aerosoliz ed virions and aggregates load onto the filter, it is possible for them to form a wide size range of superaggregates. Fig ure 6 3H shows that grape shaped superaggregates were observed in the absence of mucin. Multivalent cations of t he soluble salts (Mg 2+ an d Ca 2+ ) can interact with negative ly charge d features on the surface of MS2 to promote a high degree of virus aggregation (Sjogren and Sierka, 1994). E ncasement by a thin layer through the crosslinking network (Fig ure 6 3F) appears to result

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111 from g el forma tion caused by the presence of mucin T he similarity of the underlying structures in Fig ures 6 3F and 6 3H suggests that mucin contributes little or nothing to the aggregation process and simply covers the final configuration To isolate the UV protection effect of water insoluble mucin in AS, the IEs of MS2 nebulized in mucin free AS medium and mucin medium were investigated. For fair comparison, 0.3% and 0.6% of volume fractions w ere considered. As shown in Fig ure 6 4, for volume fraction of 0.3% the log IEs in 0.3% mucin free AS were 3.66, 4.33 and 4.94 after 30 60 and 120 min irradiation, respectively, whereas the log IEs in 0.3% mucin medium were 3.12, 3.94, and 4.37 after 30 60 and 120 min irradiation, respectively. The lower log IEs in mucin free AS compared to those in DI water suggest a protective effect of water soluble salts The higher IE in 0.6% mucin free AS compared to 0.3% mucin free AS was expected because salts increase hydration and then more water can shield viruses. Contrary to t hat expectation, the difference between 0.3% and 0.6% mucin free AS was not significant. The log IEs in 0.3% mucin free AS were higher than those in 0.3% mucin medium (salt free AS) indicating better protection by water insoluble mucin than by various wat er soluble salts. Relatively higher IEs in both mucin free AS and salt free AS than those in AS suggest that both encasement by water insoluble mucin and aggregation by water soluble salts contribute protection. In addition, that the encasement in BE provi ded better protection than in AS even through the volume fraction of solid in BE is only half of that in AS appears to indicate that the organic solids in BE are stronger absorbers at 254 nm and thus provide more effective protection from UV radiation Eff ect of RH during Bot h L oading and In ctivation Log IE s measured after aerosol loading for 30 min follow ed by UV exposure f or 60 min at different RHs are displayed in Fig ure 6 5; the corresponding general factor ANOVA

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112 results appear in Table 6 1 Because th e strong effect of the spray medium (> 80% contribution, not shown) ma de it difficult to distinguish the RH effect, two way ANOVA was also conducted for each spray medium Referring to Fig ure 6 5 upon completion of the experiment, the highest inactivation efficiency, around log 5.8, was seen in filters subjected to UV after applying the MS2 in DI water at LRH However, it should be noted that the actual IE at this condition might be somewhat higher because values measured under LRH during UV inactivation a fter aerosol loading in both LRH and MRH conditions were at the detection limit of our experimental system. For MS2 delivered in DI water, b oth RH during UV inactivation ( p < 0.0001) and RH during aerosol loading ( p < 0.0001) are significant, as is the in teraction for both RHs ( p = 0. 0118). This may be attributed to a combination of intrinsic susceptibility of MS2 and UV exposure susceptibility of MS2, augmented by stres s imposed on MS2 by aerosolization under different loading RHs. The second susceptibili ty was the more important parameter because the contribution of RH during inactivation (75%, not shown) was five times that of RH during aerosol loading (15%, not shown). In general IEs at L RH were higher than those at both MRH and HRH suggesting a prote cti ve contribution by a water layer This is broadly consistent with a report that inactivation efficiency of UV against microbes dramatically dropped off above 70% RH (Burgener, 2006). Unlike in DI water, IEs in BE were not significantly influenced by RH during virus loading, during UV irradiation for 60 min, or by their interaction IE was in the range of 2. 4 2.8 logs under all nine sets of RH conditions The contribution s of both RH regime s are less than 6% although the contribution of RH during inactiv ation is 1.9 times t hat of RH during virus loading, suggesting som e effect of water on protection by the solid content. To investigate the protective effect of solid contents in BE directly, intensities of a 1.0 mW/cm 2 UV beam after penetrating BE solution s of different concentrations were compared. Values

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113 of 0.97, 0.78, 0.62 and 0.41 mW/cm 2 were found after penetration of solutions containing 0%, 0.1%, 0.3%, and 0.5% BE, respectively, verify ing the conclusion above that UV absorbers in BE contribute signif icantly to its observed protective effect. In AS, IEs fell in the range 2.7 3.2 logs under all conditions, showing that dissolved solids in these experiments eliminated s ensitivity of the MS2 particles to RH during loading. However, RH during inactivation was a statistically significant factor ( p = 0.0204). To distinguish the significance of the RH levels, a Tukey comparison was conducted and a difference was identified at HRH. Compared to BE, AS was less protective even though the solid fraction of AS is larger and t he contribution of RH at both stages was larg er (38%, not shown) in AS than in BE (6%, not shown). These are consistent with conclusions above from SEM images and UV absorption results that the two solid media act by different routes: solids in BE appear to encase the virions in a shell that provides environmental protection and some UV screening. In contrast multivalent cations in AS appear to gather virions and promote formation of superclusters that are coated with a layer of mucin as a gel, which affords less protectivity and is more sensitive to water than the BE shell Virus Susceptibility When microbes are exposed to a biocidal factor, first order decay of viability is commonly observed (Brickner et al., 2003), and the IE of UV irradiation as a function of time can be defined as : (6 5) where A is the fraction of the total initial population subject to fast decay, N s is the concentration of airborne virus surviving after UV exposure, N o is the concentration of airborn e virus before UV exposures, C is the UV intensity factor (W/m 2 ) t is time (s) and K is the virus susceptibility factor (m 2 /J)

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114 Although this equation gives a straight line in a semi logarithmic representation, two characteristics at the begi n ning and e nd (e.g., shoulder (IE < 90%) and tailing) w ere not incorporated (Kowalski and Bahnfleth, 2000; Brickner et al., 2003). The shoulder represents the threshold dose; if the dose is insufficient, the virus shows negligible response or even recovers from the d amage. Meanwhile, the slow decay curve at tailing might be from a resistant minority of viruses and/or reaching the detection limit (Kowalski & Bahnfleth, 2000). Table 6 2 lists first order decay K s derived from the experimental results. A h igher K was ob served for both loading and exposure in LRH. In addition, and as expected, K at LRH in DI water was higher (by more than 10 x) than in AS or BE Reported UV susceptibilit ies of some other viruses are list ed in Table 6 3. K for viruses is in the range 0.01 10. The value of MS2 in DI water is similar to that of corona virus, which is of the same genom ic type. Low K values for double stran ded and DNA type viruses were expected both because the ir undamaged strand s are able to repair UV damaged segments and be cause RNA is a stronger UV absor ber than DNA (Kowalski, 2009). However, for dsDNA type v alues of K were found to vary widely depending on the individual characteristics of viruses, rather than following a simple classification by genome type. Summary T h is study examined t he effect of transmission mode and environmental conditions on sensitivity of MS2 coli phage to UV disinfection. IE was lower following droplet transmission than aerosol transmission, largely owing to the higher water content of the larg er droplets, which shields viruses from UV exposure. Lower K s were observed for viruses in BE and AS than in DI water, which is attributed to protective effects exerted by solids present in the respective media. In these experiments BE was slightly more pr otective than AS and acted by some different mechanisms. The protective effect of solids followed the order water

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115 insoluble solid (beef extract powder) > water insoluble viscous solid (mucin) > water soluble solid (salts). When these solids were present R H was not a significant parameter in decontamination by UV exposure. If the susceptibility factor is obtained for the target microbial species, the appropriate UV dose for surface decontamination can be determined.

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116 Table 6 1 Sta tistics in general factor Analysis of Variance s Factors p value Three factor ANOVA for three media spray medium < 0.0001 RH during UV inactivation 0.0197 RH during aerosol loading < 0.0001 Spray RH during aerosol loading < 0.0001 T wo way ANOVA for DI water RH during aerosol loading < 0.0001 RH durin g UV inactiation < 0.0001 RH during UV inactivation RH during aerosol loading 0.0118 Two way ANOVA for beef extract RH during aerosol loading 0.2202 RH during UV inactivation 0.4188 RH during UV inactivation RH during aerosol loading 0.6278 Two way ANOVA for artificial saliva RH during aerosol loading 0.4569 RH during UV inactivation 0.0204 RH during UV inactivation RH during aerosol lo ading 0.9983 SS: sum of squares DF: degree of freedom MS: mean square Significant parameter

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117 Table 6 2 Virus susceptibility factor K (m 2 /J) for aerosol transmission under different conditions RH condition K in Loading UV expo sure DI wate r AS BE LRH LRH 0.764 0.056 0.046 MRH 0.267 0.043 0.040 HRH 0.273 0.045 0.039 MRH LRH 0.222 0.061 0.051 MRH 0.189 0.058 0.044 HRH 0.209 0.051 0.041 HRH LRH 0.231 0.055 0.042 MRH 0.222 0.056 0.044 HRH 0.207 0.044 0.038 Table 6 3 Virus susceptibility factors (m 2 /J) from other studies Test microbe Type < 68%RH >75% RH Reference Adenovirus dsDNA 0.039 0.068 Walker & Ko (2007) Vaccinia dsDNA 6.01 1.42 McDevitt et al (2007) Phage T7 dsDNA 0.33 0.43 0.22 Tseng & Li (2005) Phage Phi 6 dsRNA 0.31 Tseng & Li (2005) Phage phi X174 ssDNA 0.71 0.38 0.53 Tseng & Li (2005) Corona virus ssRNA Walker & Ko (2007)

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118 (A) (B) F ig ure 6 1. Schematic diagrams: A) droplet loading system and B) aerosol loading system

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119 Figure 6 2. Log inactivation efficiency by UV exposure at HRH for droplet and aerosol transmission mode as a function of UV irradiation time in different nebulizer media at HRH during virus loading

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120 Figure 6 3. The SEM images of the filter contam inated with viruses aerosolized: A) and B) DI water, C) and D) 0.3% beef extract, E) and F) artificial saliva, and G) and H) artificial saliva without muc us under HRH. Magnification of A ), C), E), and G) 3,000 and B), D), (F), and H) 30, 000.

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121 Figure 6 4 Log IE after virus loading and UV exposure at HRH for aerosol transmission mode as a function of UV irradiation time in 0.3% and 0.6% mucin free ar tificial saliva and 0.3% salt free artificial saliva

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122 Figure 6 5 Log natural decay and inactivation efficiency as a function of relative humidity during b oth loading and UV inactivation: A) DI water, B) 0.3% beef extract, and C) artificial saliva.

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123 CHAPT ER 7 EFFECTS OF RELATIVE HUMIDITIES AND SPRAY MEDIA ON SURVIABILITY OF VIRAL AEROSOLS Background Viral aerosols, such as smallpox virus, SARS virus, and influenza virus, have been identified as the most common cause of respiratory infectious diseases acqu ired from recent outbreaks ( CDRF, 2006; CDC, 2009). Because they are related to the severe pandemic even ts causing death (CDC, 2009), the public spurred and their interest in the protection device such as a respirator and inactivation technology like ultraviolet germicidal irradiation (UVGI) to prevent their spread has increased. The performance of the protection and decontamination is determined by several factors related to the characteristics of viruses. For example, filtration efficiency is dependent on the size distribution of aerosols containing viruses (Hinds, 1999) and inactivation efficiency of UVGI is determined by relative humidity (RH) and media related to susceptibility of viral aerosol as well as intensity and exposure time of UV (Woo et al., Submitted). To improve the efficacy of filtration and inactivation technology, understanding the characteristics of viral aerosol is of seminal importance. The naked virion ranges from 20 nm to 300 nm in diameter but viruses in natural syste m exist in various size ranges from ultrafine (< 0.1 m), submicron (< 1 m), to super micron (> 1 m) because of aggregation of several viruses, attachment of viruses onto other airborne particles, or encasement of viru ses by droplets of respiratory secretions Inhaled particles can de posit in various respiratory regions. After they are deposited, the aggregates may disperse into numerous individual virions. More than 400 different viruses with different lethal doses (LD s) result in human diseases such as rubella, influenza, measles, mumps, smallpox, and pneumonia, which involve the respiratory system either directly or indirectly (Prescott et al., 2003). Some viruses cause disease s with

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124 only few lethal doses For instanc e, e bola virus ( = 30 nm) with a lethal dose of 10 100, results in e bola hemorrhagic fever with fatality in humans ranging from 50 89% (Biosafety level 4) ( Brion and Silverstein 1999 ). If one aggregate of e bola viruses is released from a filter surface a nd inhaled into the respiratory system as a 300 nm particle, the viruses may disperse in the pulmonary fluid and the final dose of 1000 can exceed well over the lethal level The aggregation, attachment, and encasement of viruses also facilitate resistance to environmental stresses, because of shielding effect ( Kowalski & Bahnfleth, 2007 ) and different stability at each condition L ipid viruses are stable at lower RH, where as non lipid viruses are stable at higher RH (Benbough, 1971). In addition, virus es l ose their viabilities in the presence of a N aCl or peptone containing medium whereas phenylanine protects a virus from inactivation at various RH levels ( Dubovi & Akers, 1970; Trouwborst & de Jong, 1973). Tseng and Li (2005) demonstrated that both the mo rphology of the virus particles and the presence or absence of a lipid envelope significantly affected the collection efficiency of four bioaerosol sample rs and the viability of collected virus sample. The purpose of this study wa s to investigate the envi ronmental condition like spray medium and RH on the stability for virus. By comparing the infectio us viruses obtained by plaque assay with total viruses obtained from polymerase chain reaction (PCR), the stability factor was determined for different enviro nmental conditions Such information can provide a very useful tool in designing an effective strategy to improve filtration and inactivation efficiency and for assessing the risk imposed by respiratory deposition of viral aerosol. Materials and Methods T est V irus and S praying M edium As a viral simulant, MS2 bacteriophage (MS2) was selected after considering se veral factors including eas e economics, biosafety level, size, shape, host, and available molecular

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125 techniques. MS2 ( = 27 nm) is an icosahedr al non envelope d, linear ss RNA genome virus which only infects Escherichia col i ( E. coli ) strain C3000. In addition, it contains four assembly protein), and can be ea sily analyzed by PCR and e nzyme linked immunosorbent assay ( ELISA ) It is commonly used as a non pathogenic surrogate to human pathogenic viruses (e.g., rotavirus, influenza, poliovirus, and rhinovirus ) ( Brion & Silverstiein, 1999 ). The MS2 virus stock was prepared by suspending freeze dried MS2 (ATCC 15597 a small amount of milk proteins and organic molecules for virus preservation, in filtered deionized ( DI ) water to a titer of 10 11 10 1 2 plaque forming units (PFU ) /mL and stored at 4 C. A single layer bioassay was used to enumerate the infectious viruses with E coli (ATCC 15597) as the host The details of the assay are described in Woo et al (2011). Three types of spraying media (i.e., DI water, 0.3 vol% beef extract ( BE ) and art ificial saliva ( AS )) were selected. DI water was included to explore properties of the naked virus whereas BE was used to provide a source of protein which can also contribute encasement and AS was used to mimic human respiratory fluid. The details of AS composition were reported in Woo et al. (2010). For fair comparison of protein effect between BE and AS, 0.3 vol% was selected because the concentration of the protein component (mucin) in AS was 0.3 vol%. Experimental D esign and T asks The schematic of t he experimental set up is shown in Figure 7 1. Four tasks of experiments (i.e., collection efficiency of BioSampler (Task 1), the particle size distribution (Task 2), plaque assay (Task 3), and PCR analysis (Task 4)) were performed in this study. For T ask 1, PSL particles (Duke Scientific ) with different size s from 30 nm to 300 nm were used to determine the collection efficiency of BioSampler at the specific size and flow rate.

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126 As displayed in Figure 7 1A, a six jet Collison nebulizer (BGI Inc., CN25) was u sed to generate a erosols containing viruses with a flow rate of 6 Lpm. The second dry air was added to the mixing chamber to achieve LRH in the system and then rejoined the aerosol flow. After penetrating the mixing chamber, the aerosols in the combined fl ow were collected in the BioSampler (SKC Inc., Eighty Four, PA, USA ) containing 15 mL of DI water at different flow rate s T h e collection efficiency of the BioSampler is defined as: (7 1) where N dp, down an d N dp, up are the numbe r concentrations of particles collected by condensation particle counter (CPC) at specific particles size (d p ) downstream and upstream of the BioSampler respectively. For T asks 2 4, the MS2 in three different spraying medi a (i.e., DI water, 0.3 % BE and AS ) was used instead of PSL particles in DI water. To elucidate RH effect on the stability of viruses, three RHs ( ( i.e., low RH (LRH, 30 5 %), medium RH (MRH, 60 5 %) and high RH (HRH, 90 5 % )) were applied Task 2 experi ments were performed to measure the number, surface, and mass based particle size distributions of viral aerosol by using scanning mobility particle sizer (SMPS) whereas T ask 3 experiments were to obtain size distribution of the infectious viruses at speci fic sizes by using plaque assay. The experimental set ups for T ask s 2 4 is illustrated in Figure 7 1B. For T ask 3, by adjusting the applied voltage on the DMA, only particles of the co rresponding size can penetrate the electrostatic classifier. Seven speci fic sizes from 30 nm, which is related to the MS2 v irion, to 230 nm, which is close to the upper limited particle size measured by the SMPS at a flow rate of 1.5 Lpm and 5 more sizes (i.e., 60, 90, 120, 150, and 180 nm) between them were studied The size classified particle s were collected by the BioSamp l er To minimize any loss in collected viruses over long sampling time and reaerosolization, a flow rate of 4.5 Lpm and sampling time of 5 mins

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127 were applied ( Riemenschneider et al., 2010) T h e number of via ble viruses collected in BioSample r s was enumerated by using the single layer bioassay. The size distribution function of infectious viruses based on the results of the plaque assay was calculated following Eq uation 7 2 (7 2) wh ere C PFU is the virus concentration in the collection medium of the BioSampler, V is the volume of the collection medium of the BioSampler, C Eff is the correction factor for the collection efficiency of the BioSampler for specific particle size from Task 1 Q inlet is the inlet flow rate of DMA, t is the collection time of the BioSampler, and log d p is logarithm of the bin size of the DMA. The number of MS2 PFU per particle ( N PFU ) was determined by dividing the C PFU by the total aerosol particles and t he the oretical N PFU was calculated with the volume fractio n of MS2 in the solid content in the spray medium for the given particle size, according to Eq uation 7 3 (7 3) where V dp is the volume of the droplet nulei F MS2 is the volume fraction of infectious viruses obtained from the plaque assay for MS2 stock suspension, and d MS2 is the size of MS2 virion (27 nm). Based on the composition of the media, the volume fractions of solids in DI water BE and AS were 1 10 4 3. 1 10 3 and 6 0 10 3 respectively T h e corresponding particle size of MS2 aerosols generated were 0 046 d d 0.1 4 5 d d and 0. 1 79 d d respectively when d d is the diameter of the droplet.

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128 The liquid sample assayed in Task 3 was also used for PCR to determine the concentr ation of total viruses including infectious and non infectious viruses with an assumption of no RNA d istortion during the test (Task 4). Before pro ceeding to the PCR, nucleic acid extraction was conducted with 4 mL samples from the BioSampler Because the concentration was insufficient to compare, this sample w as concentrated to 280 L by using an Amicon ultra centrifugal device (UFC 810096, Millipore, Bedford, MA, USA) The concentrated sample was processed using RNA extraction with a QIAamp Viral RNA min i kit (QIAGEN Inc., Valencia, CA, USA) and then the aliquots were stored at 80 C. Real time PCR assays were designed in O connell et al. (2006) a nd the sequence data for MS2 w ere obtained as a target of RNA replicase from National Center for Biotechnology I nformation (NCBI) with accession number of NC_001417. The target sequences were as follow: MS2 F and MS2 R: Forward and reverse primers binding to target sequences on the internal MS2 bacteriphage control, 80 pmol/ L each. MS2 F: 5 TGG CAC TAG CCC CTC TCC GTA TTC ACG 3 MS2 R: 5 GTA CGG GCG ACC CCA CGA TGAC 3 MS2 R OX /Probe: Taq man probe that hybridizes to a t arget sequence on the MS2 internal control 80 pmol/ L 5 R OX CAC ATC GAT AGA TCA AGG TGC CTA CAA GC BH Q2 3 In this study, primers and probe used were obtained from Applied Biosystem (ABI) E nzyme and buffers used were from the Invitrogen Superscript TM Platimium kit. One step Quantitative PCR (qPCR) was carried out on ABI 7500 under the following conditio n: incubation at 50 o C for 15 mins followed by further incubation at 95 o C for 2 mins and then 40 cycles of amplification with denaturation at 95 o C for 15 s and annealing and

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129 extension at 60 o C for 30 s acquiring on the ROX channel and FAM channel. MS2 R NA ( 165948, Roche Diagnostics, Indianapolis, IN, USA) with 5 serial dilutions and DNase RNase free water sterilized were substituted for MS2 RNA sample as the positive and negative controls By comparing the threshold cycle ( C T ) for positive control to th at for RNA samples, the total number of viruses ( both infectious and non infectious viruses) was calculated. The number of MS2 RNA per particle ( N RNA ) was then determined by dividing it by the total aerosol particles measured by the CPC following Eq uation 7 4 and then the stability factor of virus can be obtained b y comparing the N RNA /N PFU (7 4) Results and discuss i on Collection E fficiency of BioSamplers Figure 7 2 shows the collection efficiency of the BioSampler as a function of particle size. At a flow rate of 12.5 Lpm, the collection efficiency increased as particle size increased except ultrafine size (~30 nm), which is explained by the major collection mechanisms of impaction and centrifugation for large particles and diffu sion for small particles (Hinds, 1999) At 4.5 Lpm, the collection efficiency for all part icle size tested was below 15% which is presumably expressed by the reduced collection by inertia impaction and centrifugation because of low flow rate. Lin et al. ( 2000) investigated the effect of flow rate on collection efficiency of BioSampler and observed increases in collection efficiency as a function of flow rate. They used three flow rates of 8.5, 10.5, and 12.5 Lpm and large particles (0.8 and 1.0 m), suppor ting higher collection efficiency at 12.5 Lpm for large particles. Hogan et al. (2005) also noted that low collection efficiency of below 20% at the flow rate of 3.5 12.5

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130 Lpm for 25 nm particle. In addition, because liquid behavior in BioSampler wa s turbul ent beyond 8.7 Lpm, the collection efficiency of 300 nm dramatically increased after 8.7 Lpm. The data reported by Hogan et al. (2005) agree well for smaller (30 nm) and larger particles (300 nm) tested. Because a flow rate of 4.5 Lpm was employed for furt her test to avoid reaerosolization ( Riemenschneider et al., 2010) the collection efficiency of 12%, 8%, 6%, 4%, 4%, 6%, and 7% for 30, 60, 90, 120, 150, 180, and 230 nm, respectively, were applied as a correction factor for other tasks. Size D istribution of MS2 in D ifferent E nvironmental C onditions Figure 7 3 displays the number based particle size distribution for aerosols generated from MS2 suspension in DI water, BE, and AS under three RHs. As shown, t he particle size distribution MS2 aerosols had a mo de at approximately 2 7 n m, correspond ing to the size of MS2 virion, at LRH/RT T he number based particle size distribution s for aerosols generated f rom MS2 suspension in DI water slightly increased as RH increased, as well as geometric standard deviation ( GSD). This is explained by incomplete evaporation of water at higher RH condition although theoretically there was sufficient residence time for complete evaporation for three RHs. The particle size distribution of MS2 aerosol generated in BE and AS was s hifted to the larger particle size compared to that in DI water. T h e modes of MS2 aerosol generated in DI, BE, a nd AS were 28 nm, 68 nm, and 151 nm, respectively, at LRH. T h e mode shifted to the larger size according to the increase d volume fraction of spr ay medium, as explained in Hinds (1999) and Hogan et al. (2005). These values for BE and AS shifted to the smaller and larger size, respectively compared to the expectation of 84 nm and 106 nm The larger size of AS is presumably because of the intrinsic property of mucin component in AS. Mucin is a viscous glycoprotein, which is 75% carbohydrate and 25% amino acids li n ked via glycosidic bond s between N acetylgalactosamine and serine or threonine resides (Bansil et al., 1995).

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131 Hence, gel formation of muci n can readily retain the water contents and retard the evaporation of water at even LRH condition. Size Distribution of infectious MS2 The particle size distribution of infectious MS2 in DI water is displayed in Figure 7 4. To investigate the dimension of the particle size distribution of MS 2 it was compared to number and mass based particle size distributions The results showed that it followed the volumetric distribution. To verify this trend, the infectious PFU/particle was calculated and then regres sion analysis was conducted T he results are shown in Figure 7 5. When the same process was carried out for MRH and HRH, similar ly t h e slope of least square regression obtained for three RHs of around 3 was observed as seen in Table 7 1 However, at MRH, the number of infectious virus was smaller compared to LRH although total particle concentration was higher. This can be explained by the different stability on RH (Prescott et al., 2006). Different from the other two RHs, the number of infectious MS2 at 30 nm was much lower than expected indicating lower stability of virion at MRH condition The slopes of N PFU for BE and AS for three RHs were also obtained and listed in Table 7 1. The values are lower than that of DI wate r They were were between 2 and 3 in BE whereas around 2 in AS F i gure 7 6 shows the RNA of MS2 generated in DI water as a function o f particle diameter at three RHs as well as the theoretical value ( N theo RNA ) with an assumption of negligible impurity. Similar to N PFU the regression an alysis of N RNA for DI water was conducted as well as BE and AS and the results are listed in Table 7 2. The slope s of N RNA values in BE and AS were significantly lower than that of DI water It can be explained by the higher volume fraction resulting from the presence of solid contents. BE consists of insoluble solid s with hydrophobicity, causing the hy drophobic interaction with MS2 that attach MS 2 to the surface of insoluble s a l ts. T h e slope of N RNA in AS was less compared to

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132 the BE This might be explain ed by the higher volume fraction and the surfactant property of mucin. Mucin tend s to adsorb to hydrophobic surfaces via protein surface interactions while hold s water molecules via their hydrophilic oligosaccharide clusters. When MS2 was captured by mucin through hydrophobic interaction with MS2 protein the hydrophilic part enabled viruses not to stick (Shi et al., 2000). Mantle and Husar (2003) reported that p reincubation of plasmid bearing Yersinia enterocolitica with intestinal mucin s ignificantly redu ced subsequent binding of the organism to polystyrene, suggesting that mucin may mask hydrophobic adhesi o ns on the bacterial surface and make the microorganism more hydrophilic. The stability of MS2 at specific sizes at different RHs was com pared using the stability factor ( N PFU / N RNA ) as displayed in Figure 7 7. The stability of MS2 generated in DI water followed the order LRH > HRH > MRH (p< 0.001, Turkey s comparison < 0.05 for all), supporting Floyd et al. (1977). The stability factor of MS2 generally increased as a function of particle size, indicating the shielding effect of bigger particles. Indeed the stability factor of MS2 at MRH was very small at smaller size; however, the value dramatically increased at larger size. F o r BE, it should be noted that the stability factors were higher compared to these value for DI water, indicating pro tection effect of beef extract. Schaffer et al. (1976) and Benbough (1971) reported low protein content in the medium led to a sharp decrease in viability and Schaff er et al. (1976) showed that the minimum concentration of protein for virus stability was 0.1%. The slightly lower stability of MS2 in BE at LRH might be explained by stress of MS 2 by crystallization of solute (Trouwborst & de Jong, 1973; Hinds, 1999) Wit h the same context, prot ective effect of AS was confirmed with the higher stability value compared to that for solute free medium although lower stability factor was expected b ecause of a dverse effect of saliva component (Barlow & Donaldson, 1973). Woo et al. ( 20 12 submitted) reported the SEM image of super aggregates when AS was applied to

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133 generate viruses, supporting the cross linking effect of mucin. The viability of MS2 captured ins ide mucin linkage might be higher than naked MS2 T he effect of RH on s tability factor was negligible It might be explained by the holding of water molecules via their hydrophilic oligosaccharide clusters (Shi et al., 2000) There is less MS2 in a particle, resulting in lower shielding effect. Although higher volume fraction in AS was shown compared to BE, the lower survival fraction s in AS were generally observed. Because the volume contents of insoluble salts (0.3%) are the same, the possible reason might be soluble salt in AS, supported by Benbough (197 1 ) that the soluble salt content of the suspending medium was the main reason of the low viability at MRH This result suggested that AS showed bot h protective effect by insoluble mucin components and adverse effect by soluble salt components. Summary Both particle size dist ributions of infectious and total viruses in pure media without solute follow volumetric size distribution, whereas those in spray medium with solute follow lower dimension size like surface or number. Aggregation by MS2 itself and enc asement by inert salt s afford higher stability factor because of shielding effect and reduction of the air/water interface. In addition, for MS2 aerosols generated in gel formation media like artificial saliva the protection effect was observed but the effect of relative humi dity c ould not be distinguished.

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134 Table 7 1 Slope of least squares regression for N PFU as a function of particle size for different spray medium at three relative humidities. Spray medium Slope of N PFU (R 2 ) LRH MRH HRH DI wa ter 2.9 8 (0.98) 3. 2 2 (0.95) 2.83 (0.99) Beef extract 2. 4 3 (0.98) 2. 3 8 (0.9 8 ) 2. 37 (0.9 8 ) Artificial Saliva 2. 11 (0.98) 1. 9 8 (0.9 1 ) 2. 0 1 (0. 92 ) Table 7 2 Slope of least squares regression for N RNA as a function of particle si ze for different spray medium at three relative humidi ties Spray medium Slope of N RNA (R 2 ) LRH MRH HRH DI water 3.41 (0.99) 3.45 (0.99) 3.35 (0.99) Beef extract 2.37 (0.99) 2.15 (0.98) 2.03 (0.99) Artificial Saliva 1.82 (0.96) 1.83 (0.99) 1.83 (0.98)

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135 Figure 7 1 Schematic diagram of the experimental set up: A) the system used to determine t he collection efficiencies of BioSampler as a function of particle sizes (Task 1) and B) the system used to measure particle siz e distribution (Task 2) an d to evaluate viable (Task 3) and total (Task 4) viruses.

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136 Figure 7 2 Collection efficiency of BioSampler as a function of particle diameter with a sampling flow rate s of 4.5 and 12.5 Lpm

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137 Figure 7 3 Particle size distribution of MS2 aerosols generated with DI water, beef extract, and artificial saliva at three relative humidities

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138 Figure 7 4 Particle size distribution of numb er (solid) and mass (empty) based MS2 a erosols obtained from monitoring the SMPS and infectious viruses (cross) through plaque assay. Error bar indicates the standard deviation of triplicate test. Figure 7 5 The infectious MS2 per particle generated in DI water as a function of particle size at three relative humidities. Dash line represents the theoretical PFU per particl e. Error bar indicates the standard deviation of triplicate test.

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139 Figure 7 6 The MS2 RNA per particle generated in DI water as a function of particle size at three relative humidities. Dash line represents the theoretical PFU per particle. Error bar indicates the standard deviation of triplicate test

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140 Figure 7 7 Stability factors of MS2 as a function of diameter at three relative humidities : A) DI water, B) beef extract, and C) artificial saliva

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141 CHAPTER 8 CONCLUSIONS AND RECOMMENDATIONS This doctoral research has focused on inactivation of viral aerosol using various novel decontamination methods. Firstly, to properly evaluate the decontamination/inactivation technologies, a droplet/aerosol loading system, which can produce the representative human respiratory secretion s and can be applied for consistent and controlled delivery of aerosolized droplets containing viral agents was developed in order to properly evaluate and compare techniques for decontamination. Because t his system can be applied for inactivation of air/objec ts contaminated by all transmission modes, i.e., aerosol, droplet, and contact transmissions, it can be used to determine the protocol for decontamination test The DAC /DAS filter was prepared by periodate oxidation of a cellulose filter or incorporated s tarch The treated filter presented a slightly higher viable removal efficiency and a significantly lower relative survival fraction as treatment time increased. Increasing the residence time by lowering the filtration velocity resulted in a higher viable removal efficiency and lower survival fraction. The removal efficiency and relative survival fraction of the treated filter increased and decreased, respectively, with increasing relative humidity. The pressure drop of the treated filter was significantly lower than that of the untreated filter, which resulted in a higher filter quality. The DAC filter with sufficient moisture content had a higher removal efficiency, lower pressure drop, and better disinfection capability, which are all important attributes for practical biocidal applications. All biocidal filters (DAS/DAC) showed a significantly lower relative survivability than untreated filters, and the relative survivability decreased as the concentration of DAS or treatment time for DAC increased. For inactivation through m icrowave irradiation assisted HVAC filtration system t he distortion by thermal effect was identified to be the major mechanism. Both survival fraction and inactivation efficiency measures changed sharply above a threshold temperature of around 90 C and reached 2 log at 109 and 116 o C, respectively. Relative humidity is a

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142 significant parameter from 50~80 C and but it ceases to be significant above 90 C. Therefore, relative humidity is not a pivotal parameter for inactivation of vira l aerosols. The heating by microwave irradiation was a key factor for inactivation performance, the temperature can be selected if the target IE and SF were suggested with simply two equations below; log (IE) log (IE) inherent filtration = log (IE) microwa ve = 7.57 + 0.08 T log (SF) = 5.01 0.0 6 T (8 1) (8 2) Unlike microwave results, relative humidity is an important parameter for UV disinfection of filters as well as the solid component in spraying medium. H igh water content that absorbs UV and shiel ding of viruses near the center of the aggregate might be responsible for lower inactivation When protective medium is present, RH is not a significant parameter. These environmental conditions are not only import ant for disinfection technology but also f or susceptibility of virus itself. Both particle size distributions of infectious and total viruses in pure media without solute follow volumetric size distribution, whereas those in spray medium with solute follow a lower dimension size. Aggregation by M S2 itself and encasement by inert salts contribute to a higher stability factor because of shielding effect and reduction of the air/water interface. In addition, for MS2 aerosols generated in gel formation media like a rtificial saliva, the protecti ve effe ct was observed but less than inert salts. Based on the knowledge learned and experiences gained in this research, recommendations are made to help further advance the application of novel decontamination method against viral aerosols: 1) D evelop ment of an analytical model for disposition of viruses in aerosol in different spray media is recommended. T he model can be a very useful tool in designing an effective strategy to improve filtration and inactivation efficiency and for assessing the risk by respira tory deposition of viral aerosol 2) Characterization of virus

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143 between 20 230 nm size range was not enough to complete the model. To further expand the applicability of the model, the stability factor of a wider size range should be included. 3) Further st udies to elucidate the aggregation and encasement by TEM will be helpful. TEM can be taken after staining the lipid with selective concentration of dye (uranyl acetate). Although higher dye concentration allows easy observation of the morphology of MS2 it can also alter isoelectric point, resulting in different aggregation modes Hence, after determining the optimal dye concentration for MS2, the morphology by TEM should be observed. 4) Microwave incorporated HVAC ventilation system for real application sh ould be tested because the energy/cost is another impotent factor with performance. F o r real application, the consumption of cost/energy should be considered. 5) Because the relative humidity in duct is between 65 85% and the condition is sufficie nt for mo ld and fungi to grow, dialdehyde starch/cellulose is possible to be applied. Aldehyde has a biocidal effect against mold and some fungi I t will be helpful to use these filter s against duct type microbes.

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144 APPENDIX A PRELIMINARY TEST FOR DROPLET LOADING C HAMBER Methods Before building the droplet loading chamber, an experiment was conducted to verify the uniform deposition of aerosols using a small chamber, as shown in Figure A 1 600 mg/L of fluorescein was applied for evaluating the distribution of the d eposition. The fluorescein aerosols were generated by an ultrasonic nebulizer with a flowrate of 0.5 Lpm for 5 minutes. Four Lydall square filters with 1 inch length, placed in the small chamber as shown in Figure A 2 were used as the collection media. Af ter the loading, the fluorescein was extracted by 50 mL solution of 0.1 N NH 4 OH for 30 mins using a wrist action shaker. The fluorescein concentration was then analyzed by a fluorometer (Turner Fluorometer, Model 112). Determination of Operating Condition s Before and after experiments, the chamber was decontaminated by isopropyl alcohol for 30 mins. Six samples were placed onto the support on the turntable using sterile forceps. Theoretically, a titer of around 10 7 PFU/mL in the ultrasonic nebulizer with 5 min loading time should provide sufficient loading density (> 10 3 PFU/ cm 2 ). The titer was prepared by adding 0. 3 mL virus stock suspension into 3 0 mL artificial saliva. T he droplets from the ultrasonic nebulizer after passing the distributer entered the ch amber through 6 inlets. The size of generated and loaded droplets can be affected by the frequency of the ultrasonic generator and environmental conditions such as RH and temperature. For this study, the frequency of the generator was 2.4 MHz and the envir onmental conditions were 2 0 2 C and 35 5%. Low RH was chosen because the survivability of MS2 is high under this condition. After loading, the residual droplets were allowed to clear for 5 mins, and the FFR samples were taken out for extraction and assay. The optimal conditions to extract the virus from the FFRs were investigated by comparing virus counts under different agitation methods, extraction media, and extraction

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145 times. The experimental procedures were as follows: (1) a glass fiber filter (GelmanS cience, No. 61630) of 25 mm diameter was loaded with a known virus titer, and (2) applied filter was agitated to extract the viruses for a select period of time by a select agitation method after waiting for 10 mins, as listed in Table A 1 To evaluate if the viability of virus is influenced by the ultrasonic process, bioaerosols produced at different times were collected by a Biosampler and their viabilit ies w ere compared. The MS2 survival efficiency might be affected by the nebulization fluid the storage time and the extraction time. To examine the MS2 survivability in the spray medium, deionized (DI) water and artificial saliva (AS) were tested. The ultrasonic nebulizer was run with a flow rate of 1 Lpm and a loading time of 5 min. After the loading, 0. 25 M glycine solution was applied to extract the MS2 on the filter by the wrist action shaker to analyze the virus concentration. The sample extraction times were 1, 2, and 5 mins. The sample was then kept in the refrigerator for 2 days for the second anal ysis. S urvival fraction was defined as the ratio of virus concentrations after two days to the virus concentration in the extraction solution at the initial time. Results Fluorescein Test The calibration results of the fluorometer under 4 modes, shown in F igure A 3 indicate this fluorometer has a good performance for the concentration range of interest to this study. The results of deposition experiment are shown in Table A 2 which displays the reading from the fluorometer under a certain mode and then co nverted according to the calibration curve to acquire the concentration. The final concentration was averaged from the 3 modes (i.e. 1, 3, and 10). The corresponding CV was 4.69%, which satisfies the criterion of 20% and demonstrates the uniformity of t his method.

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146 Determination of Extraction Conditions In terms of the agitation method, as displayed in Figure A 4 the shaking method by the wrist action shaker ( Model 75, Burrell Scientific, Pittsburgh, PA) had 1.25, 1.54, and 1.89 times higher extraction e fficiency than vortexing, rotating, and sonication, respectively. This is different from previous research on best extraction method. Edward et al (2004) showed a similar efficiency in extracting MS2 from FFR coupons between vortexing and shaking. Kim et a l (2008) showed that shaking had twice higher efficiency tha n vortexing. However, ATCC medi um (organic matter) was used instead of 0.25M glycine and BG spores was applied in lieu of MS2 bacteriophage, respectively. In other words, the efficiency may be spe cies and medium dependent. With regard to the extraction medium, as shown in Figure A 5 glycine had the higher extraction efficiency. Hence, in this study, 0.25 M glycine was selected as the extraction buffer instead of DI water or 1X PBS There are two p ossible reasons for its higher extraction efficiency. First, glycine is a zwitterion which can combine with both cations and anions and provide more reaction sites. Second, glycine has a high ionic strength which can reduce the double layer strength on par ticle surface (Roger et al. 1993 ) Regarding the extraction time, the effect is shown in Figure A 6 Fifteen mins of extraction shows the best extraction efficiency. Less than 10 mins may not be sufficient to extract the virus efficiently while greater tha n 15 mins may cause damages to virus due to mechanical stress and long air contact time. From these results, the best condition to extract the sample is to use 25 mL of 0.25 M glycine solution in a wrist action shaker with a 10 o angle for 15 mins. Viabilit y during Ultrasonic N ebulization The impact of ultrasonic nebulization on virus viability in the nebulizer reservoir was investigated by measuring the viable counts over time. The results show no significant difference in virus viability between 0 and 30 m ins ( p =0.10) Apparently, the heat shock from

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147 ultrasonic vibration did not cause damage to the MS2 in the reservoir during droplet generation. T o determine the ultrasonic effect on virus aerosol during droplet generation t he viability of the viruses collec ted in the BioSampler after 5 and 10 mins of generation was examined. The theoretical concentration in BioSampler after 5 mins of nebulization is 3 10 5 PFU/mL when the virus titer in the reservoir is 1.0 10 7 PFU/mL. The 5 mins time weighted (0 5 and 5 10 m ins) average concentration of collected viruses in the BioSampler was around 3.2 10 5 PFU/mL, which is similar to the theoretical value. As demonstrated, the ultrasonic generator can be used to produce droplets containing viruses without adverse effects on their viability. Eff ect of S aliva on V iability As shown in Table A 3 the extraction time does not affect the survival fraction significantly. However, the survival fractions were higher when applying AS than DI water. The around 1 survival f ractions from AS also imply that MS2 concentration can still be accurately analyzed after 2 days if AS is used as the medium and the sample is kept in the refrigerator.

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148 Table A 1 Several agitation methods to extract the MS2 bacteriophage onto filter Final time : Used for comparison as shown in Figure A 4 Table A 2 Fluoroscein concentrations of 4 filters under 3 reading modes No. 1X 3X 10X Reading Con. (g/L) Reading Con. (g/L) Reading Con. (g/L) Avg. Con. (g/L) 1 8.09 585.50 23.70 589.97 51.96 586 .02 587.16 2 7.23 522.73 21.25 527.63 46.43 524.58 524.98 3 7.82 565.80 22.98 571.65 49.90 563.13 566.86 4 7.84 567.26 23.00 572.16 50.40 568.69 569.37 Table A 3. The survival fraction for two media after 48 hour of storage time (in duplicate) DI water Artificial saliva Time Concentration (PFU/mL) Survival fraction Concentration (PFU/mL) Survival fraction (mins) 0 days 2 days 0 days 2 days 1 284 223.5 0.78 260 240.5 0.93 2 283 275 0.97 290 306 1.06 5 257 225.5 0.88 295 301 1.02 Agitation Method A pplied time (mins) Final time Specific conditions Shaking 1, 2, 5, 10,15, 30,45 and 60 15 mins 10 o angle Vortexing 1, 2, 5, 10, 30, and 60 1 min 3200 rpm Rotating 1, 3, 5, 10, 30, and 60 10 mins 8 rpm Sonication 1, 5, 10, and 15 10 mins 40 KHz

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149 Figure A 1 Experimental set up for aerosol loading experiment by using a small chamber Figure A 2 Filter location in the small chamber for uniform distribution experiment

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150 Figure A 3. Calibration curves of t he fluorometer under 4 modes Figure A 4 MS2 extraction efficiency based on agitation method. The error bar represents one standard deviation

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151 Figure A 5 MS2 extraction efficiency based on extraction medium. The error bar represents one standard devia tion Figure A 6. MS2 extraction efficiency based on extraction time. The error bar represents one standard deviation

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152 APPENDIX B MICROWAVE ASSISTED PAN NANOFIBER FITRATION SYSTEM FOR VIRAL AEROSOL The static on filter MS2 inactivation tests were carrie d out under 500 W microwave power. As shown in Table B 1 in less than 90 seconds the survival dropped below 2 log inactivation These results suggest that the microwave irradiation can effectively decontaminate the filters with loaded MS2 To evaluate th e inactivation performance of microwave irradiation assisted nanofiber filtration system during in flight filtration against MS2, the experimental system as displayed in Figure 5 1 was used. The similar operating conditions were applied. Duplicate experime nts were carried out for MS2 with PAN nanofiber filter. The inactivation efficiency ( C E /C E ) for MS2 under different microwave powers and time intervals when PAN was employed are shown in Figure B 1. As shown, the inactivation effect of microwave against MS 2 is dependent on both microwave power and time. 2.4 log IE was shown with continuous microwave treatment at maximum power (500 W). However, the mechanical property of PAN filter was altered after continuous test because of high temperature, which makes pa rtially crosslinked ( x linked ) filter To avoid this phenomenon, x linked PAN having high thermal stability was employed instead of PAN filter. Without filter damage, the similar inactivation result was shown (Figure B 2). This inactivation effect for viru s is less than that of E. coli shown in Zhang et al (2010) This may be explained as MS2 having high er heat resistance compared to E. coli.

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153 Table B 1. Survival fraction of microorganisms under microwave irradiation Static on filter inactivation at 500 W Microwave irradiation time 30 s 45 s 60 s 90 s MS2 Mean 33.25% 4.14% 0.81% SD 6.34% 0.81% 0.34%

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154 Figure B 1. Log Inactivation efficiency by microwave irradiation assisted filtration system for PAN nanofiber filter as a function of microwa ve application time Figure B 2. Log Inactivation efficiency by microwave irradiation assisted filtration system for x PAN nanofiber filter as a function of microwave application tim e

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155 LIST OF REFERENCES Aps, J. K.M., & Martens, L. C. (2005). Review: The p hysiology of saliva and transfer of drugs into saliva. Forensic Sci ence 150, 119 131. Aranha Creado, H., & Brandwein, H. (1999). Application of bacteriophages as surrogates for mammalian viruses: A case for use in filter validation based on precedents and current practices in medical and environmental virology. J ournal of Pharm acy Science & Technol ogy 53, 75 82. Baron, P., Estill, C., Deye, G., Hein, M., Beard, J., Larsen, L., & Dahlstrom, G. (2008). Development of an aerosol system for uniform l y depositing Bacillus anthracis spore particles on surfaces. Aerosol Sci ence & Techno lgy 42, 159 172. BeMiller, N., & Whistler, R. ( 200 9). Starch: Chemistry and Technology New York: Elsevier s S c ience and Technology. Benbough, J. E. (1971) Some factors affecting the survival of airborne viruses. J ournal of Genetics Virology 10, 209 220. Betti, L., Trebbi, G., Lazzarato, L., Brizzi, M. Calzoni, G.L., & Marinelli, F. et al. (2004). Nonthermal microwave radiations affect the hypersensitive response of tobacco to tobac co mosaic virus. The Journal of Alternative and Complementary Medicine 10, 947 957. Brion, G. M. and Silverstein, J. (1999). Iodine disinfection of a model bacteriophage, MS2, demonstrating apparent rebound. Water Resource. 33, 169 179. Brickner, P., Vince nt, R., First, M., Nardell, E., Murry, M., & Kaufman, W. (2003). The application of ultraviolet germicidal irradiation to control transmission of airborne disease: bioterrorism countermeasure. Public Health Reports 118, 99 118. Bruck, C.W. (1991). Sterili zation of medical products ; Polyscience Publications: Morin Heights. Campanha, N.H., Pavarina, A.C., Brunetti, I.L., Vergani, C.E., Machado, A.L., Spolidorio, D.M.P. ( 2007 ) Candida albicans inactivation and cell membrane integrity damage by microwave irra diation. Mycoses 50, 140 147. CDC (2009). Fact sheet: n ovel H1N1 flu situation update. Available at < http://www.cdc.gov/h1n1flu.update.htm >, September 15, 2009. CDC ( 2011) Ancillary Respirator Informa tion. Available at < http://www.cdc.gov/niosh/npptl/topics/respirators/disp_part/RespSource3.html > March 22, 2011. CDRF (2006). Reusability of facemasks during an influenza pandemic: facing the flu, National Academies Press Washington D.C. Celandroni, F., Longo, I., Tosoratti, N., Giannessi, F., Ghelardi, E., & Salvetti, S., et al. ( 2004 ) Effect of microwave radiation on Bacillus subtilis spores. Journal of Applie d Microbiology 97, 1220 1227

PAGE 156

156 Chuaybamroon, P., Thunyasirinon, C., Supothina, S., Sribenjalux, P., & Wu, C. Y (201 1 ) Performance of photocatalytic lams on reduction of culturable airborne microorganism concentration. Chemosphere, 83, 730 735. Cote R. J ( 1999 ) Media composition, microbial, laboratory scale. In : Flickinger and Drew ( E d s .), Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation John Wiley & Sons, Inc. : New York pp 104 122. Damit, B., Lee, C. N, & Wu, C. Y. (2011) Flash infrared radiation disinfection of fibrous filters contaminated with bioaerosols. Journal of Applied Microbiology 110, 1074 1084. Diaz Arnold, A. M., & Marek, C. A. (2002). The impact of saliva on patient care: A literature review J ournal of Prosthetic Dentistry 88 337 343 Dodds, M. W. J., Johnson, D.A., & Yeh, C. K. (2005). Health benefit of saliva: a review J ournal of Dentistry 33 223 233 Doguid, J. P. (1946). The size and the duration of air carriage of respiratory droplets and droplet nuclei. J ournal of Hygiene 44, 471 479. Dubovi, E. J. (1971) Biological activity of the nucleic acids extracted from two aerosolized bacterial viruses. Appl ied Microbiol ogy 21, 761 762. Dubovi, E. J. and Akers, T. G. (1970) Airborne stability of tailless bacterial viruses S 13 and MS 2. Appl ied Microbiol ogy 19, 624 628. Drazen, J. M. (2002). Smallpox and bioterrorism. The New England Journal of Medicine 346, 1262 1263. Dreyfuss, M.S. & Chipley, J.R. ( 1980 ) Comparison of effects of sublethal microwave rad iation and conventional heating on the metabolic activity of Staphylococcus aureus. Applied & Environmental Microbiology 39, 13 16 ECDC (2009). daily update Pandemic (H1N1) 2009.Available at < http://ecdc.europa.eu/en/healthtopics/Documents/091020_Influenza_AH1N1_Situatio n_Report_0900hrs.pdf > September 18, 2009 Edward, D., Man, J., Brand, P., Kaststra J., Sommerer., K., & Stone, H. et al. (2 004). Inhaling to mitigate exhaled bioaerosols. PNAS. 101, 17383 17388. EPA (1984). USEPA manual methods for virology. US Environmental Protection Agency, Research and Development 600/4 84 013, Cincinnati, OH. Favero, M.S. & Bond, W.W. (1991). Disinfectio n, sterilization, and preservation ; Lea & Febiger: Philadelphia FDA (2007) Emergency Use Authorization of Medical Products U.S. Department of Health and Human Services, Food and Drug Administration, July 2007. Available at < http://www.fda.gov/RegulatoryInformation/Guidances/ucm125127.ht ml > May 3, 2008

PAGE 157

157 Feather, G. & Chen, B. (2003). Design and use of a settling chamber for sampler evaluation under calm air conditions. Aeros ol Sci ence & Techno logy 37, 261 270. Fiegel, J., Clarke, R., & Edwards, D. A. (2006). Airborne infectious disease and the suppression of pulmonary bioaerosols. Drug Discov ery Today 11, 51 57. Fisher, E., Rengasamy, S., Viscusi, D., Vo, E. & Shaffer, R. (20 09). Development of a test system to apply virus contain in g particles to filtering facepiece respirators for the evaluation of decontamination procedure. Appl ied & Environ mental Microbio l ogy 75, 1500 1507 Fisher E M Williams J L & Shaffer R E ( 20 11 ). Evaluation of Microwave Steam Bags for the Decontamination of Filtering Facepiece Respirators. PLoS ONE 110 : e18585 Floyd, R., & Sharp, D. G. (1977). Aggregation of poliovirus and reovirus by dilution in water. Appl ied & Environ mental Microb iology 33 159 166. Gabbay, J., Borkow, G., Mishal, J., Magen, E., Zatcoff, R., & Shemer Avni, Y. (2006). Copper oxide impregnated textiles with potent biocidal activities. Journal of Industrial Textile 35, 323 335. Goldblith, S.A. & Wang, D.I.C. (1967). Effect of microwaves on Escherichia coli and Bacillus subtilis. Applied Microbiology 15, 1371 1375. Gowda N Trieff N ., & Stanton J ( 1981 ). Inactivation of Poliovirus by Chloramine T. Appl ied & Environ mental Microbiol og y 42 469 476 Grinshpun, S.A., Adhikari, A.A, Li, C., Yermakov, M., Reponen, L, Johansson, E.J. & Trunov, M. (2010). Inactivation of aerosolized viruses in continuous air flow with axial heating. Aerosol Sci ence & Technol ogy 22 1042 1048 Hou, Q.X. Liu, W. Liu, Z.H. Duan, B. & Bai, L.L. (2 008). Characteristics of antimicrobial fibers prepared with wood periodate oxycellulose. Carbohydr ogen Polym er 74, 235 240 Hamid, M., Thomas, T., El Saba, A., Stapleton, W., Sakla, A. Rahman, A. (2001). The effects of microwaves on airborne microorganism s. Journal of Microwave Power and Electromangetic Energy 36, 37 45. Heimbuch, B., Wallace, W., Kinney, K., Lum ley, A., Wu, C. Y., & Woo, M. H. et al., (2010). A Pandemic Influenza Preparedness Study: Use of energetic methods to decontaminate Filtering Fac epiece Respirators Contaminated with H1N1 Aerosols and Droplets. American Journal of Infection Control 39, 1 9. Herrera, P., Burghardt, R., Huebner, H.J., & Phillips, T.D. (2004). The efficacy of sand immobilized organoclays as filtration bed materials for bacteria. Food Microbiol ogy 21, 1 9. Hinds, W. C. (1999). Aerosol Technology New York: John Wiley and Sons, Inc. Humphrey, S. P., & Williamson, R. T. (2001). A review of saliva: Normal composition, flow and function. J ournal of P rosthetic Dentistr y 85 162 169

PAGE 158

158 Jung, J., Lee, J., & Kim S (2009). Generation of nonagglomerated airborne bacteriophage particles using an electrospray technique Analytical Chemistry 81, 2985 2990. Kettleson, E.M., Ramaswami, B., Hogan, C.J., Lee, M.H., Statyukha, G., & Biswa s, P. et al. (20 09 ). Airborne Virus Capture and Inactivation by an Electrostatic Particle Collector Env ironmental Science & Technology 43 5940 5946 Khalil, H. & Villota, R. (1989). The effect of microwave sublethal heating on the ribonucleic acids of S taphylococcus aureus J ournal of Food Prot ection 52 544 548. Kim, U.J. & Kuga, S. (2004). Reactive interaction of aromatic amines with dialdehyde cellulose gel. Cellulose. 4, 287 293 Kowalski, W. & Bahnfleth, W. (2007). UVGI design basics for air and su rface disinfection. HVAC engineering 100 111. Kowalski, W. (2009). Ultraviolet Germicidal Irradiation Handbook Springer.: New York Kujunzic, E. Matalkah, F. Howard, C. Hernandez, M. & Miller, S. (2006). UV air cleaners and upper roon air ultraviolet g ermicidal irradiation for controlling airborne bacterial and fungal spores. J ournal of Occup ational Environ mental Hyg iene 3, 536. Lang, R. (1962). Ultrasonic atomization of liquid J ournal of A coustical Soc iety America 34 6 8 Lee, I S ., Kim, H. J., Le e, D.H., Hwang, G.B. Jung, J. H. & Lee, M., et al. (20 11 ) A e rosol particle size distribution and genetic characteristic of aerosolized influenza A H1N1 virus vaccine particles. Aerosol and Air Quality R esearch 1 1 230 237 Lee, J.H., Wu, C.Y., Lee, C.N., Anw ar, D., Wysocki, K. M., & Lundgren, D A. et al. (2009). Assessment of iodine treated filter media for removal and inactivation of MS2 bacteriophage aerosols. Journal of Applied Microbiology 107, 1912 1923. Li, C.S., Hao, M.H., Lin, W.H., Chang, C.W., & Wang C. S. (1999) Evaluation of microvial samplers for bacterial microorganisms. Aerosol Sci ence & Technol ogy 30, 100 108. Lin C.Y. & Li, C.S. (2003). Inactivation of microorganisms on the photocatalytic surfaces in air. Aerosol Sci ence & Technol ogy 37, 939 944. Lin X. Reponen T. Willeke K. Wang Z. Grinshpun S.A. & Trunov M. (2000). Survival of a irborne m icroorganisms d uring s wirling a erosol c ollection Aerosol Sc ience & Technol ogy 32, 184 196 Mantle, M., & Husar, S.D. (1993). Adhesion of Yers inia enterocolitica to Purified Rabbit and Human Intestinal Mucin Infection and Immunity 61, 2340 2346 Marple, V., & Rubow, K. (1983). An aerosol chamber for instrument evaluation and calibration. Am erican Ind ustry Hyg iene Assoc iation J ournal 44, 361 367 May, K. R. (1973). The collision nebulizer: Description, performance and application. Journal of Aerosol Science 4, 235 238.

PAGE 159

159 McCrumb, F. (1961). Aerosol infection of man with Pasteurella tularensis Bacteriol ogy Review 25 1912 1923. McDevitt, J.J., Lai, K.M., Rudnick, S.N., Houseman, E.A., First, M.W., & Milton, D.K. ( 2007). Characterization of UVC light sensitivity of vaccinia virus. Applied & Environmental Microbiology 73 5760 5766 Mcdonnell, G. & Russ ell, A. D. (1999). Antiseptics and disinfectants: a ctivity, action, and resistance. Clinical Microbiology Reviews. 12, 147 179. Metcalf, E. & Eddy, F. (2004) Wastewater Engineering: Treatment and reuse ; McGraw Hill, Inc: New York Miller M.B. (2009). Removal of waterborne pathogens using an antimicrobial f ilter media, Proceeding of the 2009 Georgia Water Resources Conference Morawska, L., Johnson, G., Ristovski, Z., Hargreaves, M., Mengersen, K., Corbett, S., et al., (2009). Size distribution and sites of origin of droplets expelled from the human respirat ory tract during expiratory activities J ournal of Aerosol Sci ence 49 256 269 Munro, L. (2007) Basics for microbiology laboratory North Carolina: Contemporary publishing compamy of raleigh, Inc. NIOSH (2005). Determination of particulate filter penetra tion to test against liquid articulates for negative pressure, air purifying respirators standard testing procedure (STP). Pittsburgh, PA. O'Connell, K. P., Bucher, J. R., Anderson, P. E., Cao, C. J., Khan, A. S., Gostomski, M. V. & Valdes, J. J. (2006) Re al time fluorogenic reverse transcription PCR assays for detection of bacteriophage MS2. Appl ied & Environ mental Microbiol ogy 72 478 483. OSHA (2012). Available at < http://www.osha.gov/pls/oshaweb > Janua ry 15, 20 12 Para, A. Karolczyk Kostuch, S. & Fiedorowicz, M. (2004). Dihydrazone of dialdehyde starch and its metal complexes. Carbohydr ogen Polym er 56, 187 193. Park, D. K., Bitton, G., & Melker, R. (2006). Microbial inactivation by microwave radiation in the home environment. Journal of Environmental Health 69, 17 24. Park, J., Yoon, K., Kim, Y., Byein, J. & Hwang, J. (2009). Removal of submicron aerosol particles and bioaerosols using carbon fiber ionizer assisted fibrous medium filter media. Journal of Mechanical Science & Tech nol ogy 23, 1846 1851. Environmental Health Perspectives 102, 840 845. Perier, C., Bov, J., & Vila, M. (2011). Mitochondria and Programm ed Cell Death in Parkinson's Disease: Apoptosis and Beyond. Antioxid ant Redox Signal Jul y 18. [Epub ahead of print]

PAGE 160

160 Phelan, A.M., Neubauer, C.F., Timm, R., Neirenberg, J., & Lange, D.G. ( 1994 ) Athermal alterations in the structure of the canalicular membr ane and ATPase activity induced by thermal levels of microwave radiation. Radiation Research 137, 52 58. Power, E.G.M. & Russell, A.D. (1990). Sporicidal action of alkaline glutaraldehyde: factors influencing activity and a comparison with other aldehydes. J ournal of Appl ied Bacterio logy 69, 261 268 Prescott, L. M., Harley, J. P. & Klein, D. A. (2006) Microbiology, New York, NY: McGraw Hill Co mpanies, Inc. pp. 142 146. Radley J. A (1976). Starch Production Technology London: Applied Science Publisher, Inc. Ratnesar Shumate, S., Wu, C.Y., Wander, J., Lundgren, D., Farrah, S., Lee, J.H., et al. (2008) Evaluation of physical capture efficiency and disinfection capability of an iodinated biocidal filter medium. Aerosol Air Qual ity Res earch 8, 1 18. Rengasamy, S., Fisher, E., & Shaffer, R. (2010) Evaluatio n of the survivability of MS2 viral aerosols deposited on filtering face piece respirator sam ples incorporating antimicrobial technologies. American Journal of Infection Control 38, 9 17. Riemenschneider, L., Woo, M. H., Wu, C.Y., Lundgren, D. A., Wander, J. & Lee, J.H. et al. (2010). Characterization of reaerosolization from impingers in an eff ort to improve airborne virus sampling. Journal of Applied Microbi ology 108, 315 324. Ryan, K., McCabe, K., Clements, N., Hernandez, M., & Miller, S. (2010). Inactivation of airborne microorganisms using novel ultraviolet radiation sources in reflective fl ow through control devices. Aerosol Science & Technolog y 44, 541 550. Salgado, C. D., Farr. B. M., Hall, K. K., & Hayden, F. G. (2002). Influenza in the acute hospital setting. The Lancet Infectious Diseases 2, 145 155. Shi, L., Ardehali, R., Caldwell, K.D ., & Valint, P. (2000). Mucin coating on polymeric material surface to suppress bacterial adhesion. Colloids and Surfaces B: Biointerfaces 17 229 239 Silver, S., Phung, L. & Silver, G. (2006). Silver as biocidaes in burn and wound dressings and bacteria l resistance to silver compounds. Journal of Industrial Microbiology Biotechnology 33, 627 634. Sjogren, J.& Sierka, R. (1994). Inactivation of phage MS2 by iron aided titanium dioxide photocatalysis. Applied and Environmental Microbiology 60, 344 347. Son g, L. (2008). Antibacterial and antiviral study of dialdehyde polysaccharides. Dissertation in University of Florida Song, L. Cruz, C. Farrah, S. R. & Baney, R. H. (2009). Novel antiviral activity of dialdehyde starch. Elec tronic J ournal of Biotechnol o gy 12, 1 5. Tabak, L. A. (1995). In defense of the oral cavity: structure, biosynthesis, and function of salivary mucin. Annual Review of Physiology 7 547 564

PAGE 161

161 Tellier, R. (2006). Review of aerosol transmission of influenza. Emerg envy Infect ion Disease 12, 1657 1661. Tseng, C. & Li, C. S. (2005). Inactivation of virus containing aerosols by ultraviolet germinidal irradiation. Aerosol Science & Technolog y 39 361 366 U.S. Army (1998) Filter medium, fire resistant, high efficiency, mi litary Specification MIL F 51079D, Aberdeen Proving Ground, MD: U.S. Army Armaments Munitions and Chemical Commands. Valegard, K., Lijas, L., Fridborg, K., & Unge, T. (1990). The three dimensional structure of the bacterial virus MS2. Nature 345, 36 41. V aravinit, S., Chaokasem, N., & Shobsngob, S ( 2001 ). Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose. World Journal of Microbiology & Biotechnology 17 721 725 Veelaert, S., Devit, D., Gotlieb, K. F., & Verhe, R. (1997). The gela tion of dialdehyde starch. Carbohydrate Polymer 32, 131 139 Veerman, E. C. I., van den Keybus, P. A. M., Vissink, A., & Nieuw Amerongen, A. V. (1996). Human glandular salivas: their separate collection and analysis. Eur opean J ournal of Oral Sc ience 104, 346 352. Verdenelli, M.C. Cecchini, C. Orpianesi, C. & Dadea, G.M. (2003). A. Efficacy of antimicrobial filter treatments on microbial colonization of air panel filters. J ournal of Appl ied Microbio logy 94, 9 17 Vingerhoeds, M. H., Blijdenstein ,T. B. J ., Zoet F. D., & Aken, G. A. V. (2005). Emulsion flocculation induced by saliva and mucin. Food Hydrocolloids 19, 915 922. Vi scusi, D. F., Bergman, M. S., Eimer, B. C. & Shaffer, R. E. (2009) Evaluation of five decontamination methods for filtering facepi ece respirators. Annual Occupational Hygiene 53, 815 817. V o E., Rengasamy, S., & Shaffer, R. E. (2009) Development of a test system to evaluate procedures for decontamination of respirators containing viral droplets Applied & Environmental Microbiology 75, 7303 7309. W alker C M ., & Ko G (20 07 ). Effect of ultraviolet germicidal irradiation on viral aerosol Environmental Science & Technology 41, 5460 5465 Watanabe, K., Kakita, Y., Kashige, N., Miake, F., & Tsukiji, T. (2000). Effect of ionic strengt h on the inactivation of micro organisms by microwave irradiation. Letters in Applied Microbiology 31, 52 56. Williamson, K., Wommack, K., & Radosevich, M. (2003). Sampling natural viral communities from soil for culture independent analyses. Appl ied & En viron mental Microbio logy 69, 6628 6633. Willeke, K., Lin, X. & Grinshpun, A. S. (1998) Improved aerosol collection by combined impaction and centrifugal motion. Aerosol Sci ence & Technol ogy 28 439 456

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162 Wong, L., & Sissions, C. H. (2001). A comparison of h uman dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva. Oral Biol ogy 46, 477 486. Woo, I. S., Rhee, I. K., & Park, H. D. ( 2000 ) Differential damage in bacterial cells by microwave radiation on the ba sis of cell wall structure. Applied & Environmental Microbiology 66, 2243 2247. Woo, M H., Hsu, Y. M., Wu, C. Y., Heimbuch, B. & Wander, J. (2010). Method for contamination of filtering facepiece respirators by deposition of MS2 viral aerosol. J ournal of Aerosol Sci ence 41, 944 952. Woo, M H., Lee, J. H., Rho, S. G., Ulmer, K., Welch, J., & Wu, C. Y. (201 1 ). Evaluation of the performance of dialdehyde cellulose filters against airborne and waterborne bacteria and viruses Industrial & Engineering Chemist ry Research 50 11636 11643 Woo, M. H., Anwar, D., Smith, T., Grippin, A., Wu, C. Y., & Wander, J. (2012). Investigating the effects of relative humidities and nebulized media on UV inactivation of viral aerosols loaded filter. Submitted to Applied & Envi ronmental Engineering. Wu, Y. & Yao, M. (2010 a ). Inactivation of bacteria and fungus aerosols using microwave irradiation. Journal of Aerosol Science 41, 682 693. Wu, Y. & Yao, M. (2010 b ). Effects of microwave irradiation on concentration, diversity and ge ne mutation of culturable airborne microorganisms of inhalable sizes in different environments Journal of Aerosol Science 4 2 800 810 Yang, S. H., Lee, G. W. M., Chen, C. M., Wu, C. C., & Yu, K. P. (2007). The size and concentration of droplets generate d by coughing in human subjects. J ournal of Aerosol Med icine 20, 484 494. Yu, J., Chang, P., & Ma, X (2010). THe preparation and properties of dialdehyde starch and thermoplastic dialdehyde starch Carbohydrate Polymer 79 296 300 Zhang, Q., Damit, B., W lech, J., Park, H., Wu, C. Y., & Sigm u nd, W. (2010). Microwave assisted nanofibriuos air filtration for disinfection of bioaerosols. Journal of Aerosol Science 41, 880 888.

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163 BIOGRAPHICAL SKETCH Myung Heui Woo was born in Mokpo South Korea in 19 8 0, and was raised through h er high school years there. Sh e attended the Department of Chemistry in Korea University and received Summa cum laude for her Bachelor of Science in 2004 In 20 06 sh e received her m aster s degree in physical chemistry from K orea Universit y. She served as a graduate research assistant and teaching assistant, involved in projects titled Zirconium Phosphate sulfonated poly (Fluorinated arylene ether)s composite membranes for PEMFCs at 100 140 o C In 200 7 Myung Heui Woo enrolled at Universi ty of Florida to pursue a Ph.D. in Environmental Engineering Sciences and served as a research and teaching assistant.