PAGE 1 1 ASSESSMENT OF THE PERFORMANCE OF IODINE-TREATED BIOCIDAL FILTERS AND CHARACTERIZATION OF VIRUS AEROSOLS By JIN-HWA LEE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 PAGE 2 2 2009 Jin-Hwa Lee PAGE 3 3 To my family in Korea, my son Luke, and my husband Youn-Sung Choi PAGE 4 4 ACKNOWLEDGMENTS First and foremost I would like to gratefully and sincerely thank my advisor, Dr. Chang-Yu Wu, for his guidance throughout my graduate stu dy with constant encouragement and insightful idea. This study would not have been co mpleted without his patience and help. I would like to convey my special thanks to my committee members. First, Dr. Joseph Wander gave me consistent advices and precious ideas to improve this study. Second, Dr. Dale A. Lundgren gave me valuable comments on my study and encouragement with generosity and kindness. Further acknowledgment is extended to Dr. Samuel Farrah, Dr. Jean M. Andino, Dr. Yiider Tseng, and Dr. Ben Koopman for their in terest and suggestions on my research. It was a great pleasure to wo rk with fantastic colleagues, Alex D. Theodore, Qi Zhang, Danielle Hall, Brian Damit, and Seungo Kim, w ho work hard and have willingness to provide help at anytime. I would like to express my gr atitude to Dr. Sewon Oh, a visiting professor from Sangmyung University, for his help and advice. My special thanks go to my friends, MyungHeui Woo, Dr. Yu-Mei Hsu, Dr Ying Li, Cheng-Chuan Wang, An adi Misra, Charles Jenkins, and Lindsey Riemenschneider for their contribut ion to my study with interactive discussion, kindness that have made my study a delightful me mory. I am also grateful to undergraduate students, Katie M. Wysocki, Christiana N. L ee, Ariana N. Tuchman, Diandra Anwar, Sang-Gyou Rho, and James Welch for their techni cal assistance and priceless help. I dedicate this dissertation to my family. I would like to e xpress my deepest and sincerest gratitude to my family in Korea for their endl ess love and support. Finally, my love and many thanks go to my greatest blessing, my husba nd, Youn-Sung Choi for his encouragement and consistent support, and my son, Luke J un-Young Choi, who has grown up healthily. PAGE 5 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........7LIST OF FIGURES................................................................................................................ .........9ABSTRACT....................................................................................................................... ............11CHAPTER 1 INTRODUCTION..................................................................................................................13Biological Threat.............................................................................................................. ......13Bioaerosols.................................................................................................................... .........14Stability of Viral Aerosols.................................................................................................... ..18Polymerase Chain Reaction Analysis.....................................................................................20Filtration..................................................................................................................... ............21Iodine as a Disinfectant....................................................................................................... ...25Iodinated Resin Filter Media..................................................................................................27Research Objectives............................................................................................................ ....292 EFFICACY OF IODINE-TREATED BIOCIDAL FILTER MEDIA AGAINST BACTERIAL SPORE AEROSOLS.......................................................................................31Objective...................................................................................................................... ...........31Materials and Methods.......................................................................................................... .31Results........................................................................................................................ .............36Discussion..................................................................................................................... ..........383 ASSESSMENT OF IODINE-TREATED FILTER MEDIA FOR REMOVAL AND INACTIVATION OF MS2 BACTERIOPHAGE AEROSOLS.............................................47Objective...................................................................................................................... ...........47Materials and Methods.......................................................................................................... .47Results........................................................................................................................ .............53Discussion..................................................................................................................... ..........584 CHARACTERIZATION OF MS2 BACT ERIOPHAGE AEROSOLS INFLUENCED BY RELATIVE HUMIDITY AND SPRAY MEDIUM........................................................69Objective...................................................................................................................... ...........69Materials and Methods.......................................................................................................... .69Results and Discussion......................................................................................................... ..73 PAGE 6 6 5 CONCLUSIONS AND RECOMMENDATIONS.................................................................96APPENDIX A RAW DATA OF BACTERIAL SPORE EXPERIMENTS.................................................100B PROCEDURES FOR PREPARIN G PLAQUE ASSAY MEDIA.......................................108C RAW DATA OF VI RUS EXPERIMENT...........................................................................109D PARTICLE SIZE DI STRIBUTIONS...................................................................................121E RAW DATA OF CHARACTERIZATION EXPERIMENT...............................................124LIST OF REFERENCES.............................................................................................................127BIOGRAPHICAL SKETCH.......................................................................................................138 PAGE 7 7 LIST OF TABLES Table page 2-1 Removal efficiency of the iodine-treated and untreated filter s for bacterial spore aerosols at various environmental conditions....................................................................432-2 Survival fraction of bacterial spores on both filters at various environmental conditions..................................................................................................................... ......443-1 Removal efficiency of the iodine-treated and untreated filters for MS2 aerosols at various environmental conditions in impinge rs containing phosphate buffered saline.....633-2 The survived MS2 among various MS2 con centrations in the im pingers with various reaction solutions at various environmental conditions.....................................................643-3 Iodine concentration (mg I2/L) in the vortexing solution at each vortexing time..............653-4 Extracted fraction of MS2 on the iodine-treated and untreated filters at various environmental conditions...................................................................................................654-1 Collection efficiency of the BioSampler fo r select particle sizes adopted from Hogan et al.......................................................................................................................... ...........864-2 Components of artificial saliva..........................................................................................864-3 Slope of least squares regression line for NPFU vs. particle size for different MS2 suspensions at three relative humidities.............................................................................874-4 NRNA for MS2 aerosols generated from sterile DI water at three relative humidities........874-5 Slope of least squares regression line for NRNA vs. particle size for different MS2 suspensions at three relative humidities.............................................................................884-6 NPFU / NRNA for MS2 aerosols generated from ster ile DI water at three relative humidities..................................................................................................................... ......884-7 NRNA for MS2 aerosols generated from tryptone solution at three relative humidities......894-8 NPFU / NRNA for MS2 aerosols generated from tr yptone solution at three relative humidities..................................................................................................................... ......894-9 NRNA for MS2 aerosols generated from artificia l saliva at three relative humidities.........904-10 NPFU / NRNA for MS2 aerosols generated from ar tificial saliva at three relative humidities..................................................................................................................... ......90A-1 Impactor results at room temp erature and low relative humidity....................................100 PAGE 8 8 C-1 All glass impinger results at various environmental conditions......................................109E-1 Bioassay results (plaque-forming units)..........................................................................124E-2 Polymerase chain reaction results (ng)............................................................................126 PAGE 9 9 LIST OF FIGURES Figure page 2-1 Experimental setup for bacterial aero sol system...............................................................442-2 Particle size distributi on of entering bioaerosols...............................................................452-3 Relative fraction of spores in the vortexi ng solution of the clea n iodine-treated and untreated filters.............................................................................................................. ....452-4 SEM images of the filters at 100X.....................................................................................463-1 Experimental set-up........................................................................................................ ...663-2 The number-based particle size distributi on of aerosols entering and penetrating the filter at RT/LRH............................................................................................................... ..673-3 The survived MS2 aerosols among penetrat ed MS2 aerosols from the iodine-treated filter with various reaction solutions as the collection medium of the impinger...............673-4 SEM images of the filter at 2700X....................................................................................684-1 Conceptual schematic of the experimental set-up.............................................................914-2 Particle size distribution of MS2 aerosols generated from sterile DI water at low relative humidity.............................................................................................................. ..924-3 NPFU for MS2 aerosols generated from sterile DI water at three relative humidities........924-4 Particle size distribution of MS2 aerosols generated fr om tryptone solution at low relative humidity.............................................................................................................. ..934-5 NPFU for MS2 aerosols generated from tryptone solution at three relative humidities......934-6 Particle size distribution of MS2 aerosols generated fr om artificial saliva at low relative humidity.............................................................................................................. ..944-7 NPFU for MS2 aerosols generated from artific ial saliva at three relative humidities.........944-8 Theoretical droplet nuclei diameter as a function of dr oplet diameter for different nebulizer suspnesions at low relative humidity.................................................................95D-1 Particle size distribution of MS2 aerosols generated from sterile DI water at medium relative humidity..............................................................................................................121D-2 Particle size distribution of MS2 aerosols generated from sterile DI water at high relative humidity..............................................................................................................121 PAGE 10 10 D-3 Particle size distributi on of MS2 aerosols generated from tryptone solution at medium relative humidity................................................................................................122D-4 Particle size distribution of MS2 aerosols generated fr om tryptone solution at high relative humidity..............................................................................................................122D-5 Particle size distribution of MS2 aerosols generated from artificial saliva at medium relative humidity..............................................................................................................123D-6 Particle size distribution of MS2 aerosols generated fr om artificial saliva at high relative humidity..............................................................................................................123 PAGE 11 11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ASSESSMENT OF THE PERFORMANCE OF IODINE-TREATED BIOCIDAL FILTERS AND CHARACTERIZATION OF VIRUS AEROSOL By Jin-Hwa Lee August 2009 Chair: Chang-Yu Wu Major: Environmental Engineering Sciences The increasing threat of biological warfare a nd the spread of airborne pathogens have attracted the publics attention to bioaerosols and the need for the development of better methods for respiratory protection. Among biological agen ts, spores and viruses are of special concern because of resistance to inactivat ion treatment, small particle si ze and low infectious dose. In this study, the performance of an iodine-treated biocidal filter combining mechanical filtration and disinfection property of iodine was evaluated for Bacillus subtilis spores and MS2 bacteriophage as a surrogate for human pathogenic biological agents. Furthermore, the fate of viral aerosols influenced by envi ronmental conditions and the spra y medium were investigated by assessing infectious and non-in fectious MS2 as a function of particle size with bioassay and polymerase chain reaction. The iodine-treated filter has an excellent filt ration efficiency for bacterial spores with a negligible pressure drop in various environmen tal conditions. Inactivation of the collected spores is only slightly enhanced by the presence of the iodinate d resin. In the viral aerosol experiment, the iodine-treated fi lter also showed high biocidal performance. Both dissociation and capture iodine by viral aerosols traversing the filter are mechanisms responsible for the PAGE 12 12 inactivation. Significantly low pressure drop along with high viable removal efficiency imply its promising application as a respiratory protection device. The strong retention capability of the electrets filter minimizes reaeroslization but also makes it difficult to discriminate the antimicrobial effect at the surface. The distribution of infectious MS2 aerosols follows volume-based size distribution for relatively pure viral aerosols; meanwhile, solid-containing viral aerosols follow a dimension dependence of lower size. Enumeration of infect ious MS2 virions increa ses as relative humidity (RH) decreases and particle size increases owing to greater contribution of MS2 to the particle content. MS2 aerosols present stability at low RH s, while they are susceptible at higher RHs due possibly to the increased air/water interface. A ggregation results in shie lding effect and inert constituents yield an encasement effect because of reduced cont act to the air/water interface. However, for MS2 aerosols generated with artific ial saliva, these protective effects cannot be distinguished. PAGE 13 13 CHAPTER 1 INTRODUCTION Biological Threat The perceived threat of bioterrorism afte r the anthrax attack on September 18, 2001 and airborne virus outbreaks, includ ing historical epidemics of in fluenza, occurrences of SARS (severe acute respiratory syndrome), avian flu vi ruses, and more recently influenza A (H1N1), have drawn public attention to bi oaerosols and protection methods. Biological agents have been used throughout the history as a weapon. In the 6th century B.C., Assyrian s poisoned the wells of their enemies with rye ergot. In 1995, Aum Shin rikyo attempted on several occasions to release biological agents such as anthrax, botulinum toxi n and ebola in aerosol fo rm. Biological warfare agents can be made even by small groups and te rrorist organizations b ecause the production of bacteria, massive toxins and virule nt strains of virus is easy and in expensive. They can be more fatal threat than chemical weapons; a few kil ograms of anthrax can kill as many people as a Hiroshima-size nuclear bomb (Prescott et al. 2002 ). Being invisible, odorless, and tasteless, biological agents can spread and remain undetect ed until symptoms are developed by infected people. Biological agents can be spread widely throughout a city or re gion, in contrast to chemical agents, which spread narrowly in downw ind area near the point of release (Henderson 1999). Bacillus anthracis, one of the agents of concern list ed by the Centers for Disease Control and Prevention (CDC), was used as a bioterro rism weapon in 2001 resulting in five deaths among the 11 people known to have inhaled it. Approximately 60 million dollars were spent to provide medical treatment to affected workers a nd to test and clean up the facility. It also resulted in the launching of Public Health Security and Biote rrorism Preparation and Response Act of 2002 by the US government (MIPT 2002). PAGE 14 14 The spread of airborne pathoge ns is another emerging problem that increases the publics awareness of bioaerosols. For instance, SARS a viral respiratory illness is caused by a corona virus for which there is no vaccine. First reporte d in Asia in February 2003, SARS spread to more than two dozen countries in North America, South America, Europe and Asia over the following few months and resulted in deaths of 774 people amongst the total of 8,098 people infected worldwide. In additi on, the more recent outbreak of in fluenza A (H1N1) virus sparked fears of a pandemic sweeping the world. Like SARS virus, there is currently no vaccine for the H1N1 virus and it is expected that people do not have immunity to this new virus (CDC 2009). Transmission of SARS and H1N1 viruses are susp ected to occur through dr oplets generated from sneezing or coughing of an infected person, which s ubsequently deposit on or are transferred to the mucous membrane of the mouth, nose or eyes of nearby persons (CDC 2005). Besides these viruses, infection transmitted by the respirator y route include tubercul osis, mumps, measles, pneumonia, influenza, any many diseases not know n to humans (Biswas a nd Wu 2005; Fiegel et al. 2006). Bioaerosols Even though interest in bioaer osols has recently been highlig hted, bioaerosols have been present in the environment from the origin of mankind in both indoor and outdoor air. Bioaerosols are aerosols of biological origin including viable bacteria, viruses, fungi and algae as well as such nonviable materials as dust mi tes, pollen, endotoxins, my cotoxins and various allergens (Hinds 1999a). The size of bioaer osols ranges from aerodynamic diameters smaller than 0.5 m to 100 m (Cox 1995). Although the size of a single bacterium is commonly around 1 m with various shapes such as sp heres (cocci), rods (bacilli) or spirals, they are present in larger sizes as clusters or chains (Hinds 1999a). Larger bioaerosols are influenced by PAGE 15 15 gravitational force and are removed from air by se ttling in a short period of time. In contrast, smaller bioaerosols can remain in the air for a pr olonged period of time and travel a considerable distances by themselves or attach ed to non-biological particles ( e.g., dust) in an air. Various diseases such as t uberculosis, mumps, measles, rubella, pneumonia, meningitis, legionellosis, and influenza can be transmitted by bioaerosols (Jacoby et al. 1998). Bioaerosols need to be viable to be infectious, but viability is not a prerequisite to al lergenic and toxic effects (Baron and Willeke 2001). Non-viable bioaerosols can also cause allergic reactions by contact and inhalation (Maus et al. 2001). Biological agen ts are also correlated with building-related illness (BRI) such as legionellosis and aspergi llosis (Kemp et al. 1995b). Airborne transmission of respiratory diseases is classified into two groups: communicable and non-communicable. Communicable diseases can transmit between human hosts, while non-communicable diseases come only from the environment due to fungal or actinomycete spores and environmental or agricultural bacteria (Kow alski and Bahnfleth 1998). Most terrestrial surfaces exposed to air moveme nt can be potential sources of bioaerosols. Microorganisms in natural waters as well as anthropogenic water re main airborne after evaporation of the liquid resulti ng from rain, splashes, or bubbling processes. The growth and multiplying of microorganisms in a new environment of engineered systems such as humidifiers, evaporative air coolers, coo ling coil drain pans, and condens ation on ductwork insulation can result in an amplification of microorganisms to unhealthy levels (Kowalski and Bahnfleth 1998). Therefore, the heating, ventilation and air cond itioning (HVAC) system of a building can be a major source of bioaerosols indoors (Baron and Willeke 2001). Workers in occupational environments where organic materials such as pl ants, hay, organic waste, wastewater, cotton and metalworking fluids are handled are exposed to high concentrations of bioaerosols. Through PAGE 16 16 sneezing and coughing, humans are al so one of the most important sources of bioaerosols. Specifically, a single sneeze can generate a hundred thousand bioaerosols. A single cough produces only one percent of this amount, but 10 times more frequently th an sneezes (Kowalski and Bahnfleth 1998). Thousands of droplets approximately 1 to 10 m in diameter and containing viable microorganisms released by a pe rson will quickly evaporat e to droplet nuclei. For instance, the evaporation time of a 12-m droplet is only 0.02 s. The droplet nuclei remain suspended in air for a long time and travel cons iderable distances by attaching to aerosols existing in air. Especially, respiratory viruses such as influenza virus appears to be spread mainly by droplet nuclei (Small 2002; Beggs 2003). Virus infectivity is shielded from drying, sunlight, and temperature compared to an isolat ed airborne virus due to encasement of droplet (Tyrrell 1967). In indoor envir onments, microorganisms are also free from factors inducing their destruction, thus result ing in longer survival. Direct su nlight has the po tential to kill microorganisms since it contains a lethal level of ultraviolet radiation. Oxygen and pollutants in air may also be sources of the destruction of microbes. A st udy on the loss of viability of airborne microbes revealed that in the absence of sunlight, bacteria deca y faster in air than viruses, because bacteria depend more on moisture for their survival than viruses do (Kowalski and Bahnfleth 1998). Among the various biological agents, bacterial spores and viruses are of special concern because of their unique properties. In adve rse environmental conditions, certain species of bacteria can survive by forming endospores exhi biting incredible longevity and resistance to environmental stress (Nicholson et al. 2000). Germ ination and the outgrowth of vegetative cells are initiated when the endos pores encounter an appropria te environmental trigger, e.g. a simple amino acid or riboside (Moir et al. 2002). Bacter ial spores are highly re sistant to deactivation, PAGE 17 17 such as by heat, radiation and chemical agents. Specific properties of spores are responsible for their resistance, including low water content in the core and saturation of the spore deoxyribonucleic acid (DNA) with a group of small, acid-soluble spore proteins (SASP) of the / -type (Popham et al. 1995; Tennen et al. 2000). T hus, bacterial spores have been classified as a group of bioagents for which treatment and disinfection are specially challenging. Viruses are the smallest biological agents; a single naked virus ranges from 20 300 nm. However, in the natural environment, they are no t typically present as a single naked virus due to aggregation of several singl e viruses or attachment to non-biological particles ( e.g., dust) in the air that result in several unique properties of their own (Hinds 1999a). Because of the surrounding outer layer of viruses, the inner viruses of an aggregate can be protected from inactivation treatments. This s hielding effect of viral aggregates from inactivation treatment was already observed in water (Gal asso and Sharp 1965). When viru ses are attached or enclosed to the surrounding substances in the air, their in fectivity can also be pr eserved by reduced contact with inactivation agents, i.e., an encasement effect. Sec ond, the viral aggregates can be present in the ultrafine size range Ultrafine particles are of gr eat concern because respiratory deposition of a significant number of these particle s is easily achieved. Beha viors in contrast to larger sized particles include translocation to ex trapulmonary sites and migration to other target organs by a different transfer rout e (Oberdorster et al. 2005). Inhalation of aggregated ultrafine biological agents can result in fatal consequences, because only minute amounts of viruses are needed to cause disease (Hinds 1999a). For ex ample, the infectious dose of smallpox is 10 100 viruses and that of viral hemorrhagic fever is 1 10 viruses (Pien et al. 2006). The low dose is estimated and true the infecti ous dose of many virus agents is unknown. Furthermore, a small viral particle can be susp ended in air for a long time and travel considerable distances by itself or PAGE 18 18 by attachment to a non-biological pa rticle resulting in a higher potential to sp read disease. The other concern is that virions and even viral aggr egates are in the size ra nge of minimum filtration efficiency in air (usually 0.05 0.5 m) (Hinds 1999b). Although the nominal MPPS (most penetrating particle size) de signated by the NIOSH filter ce rtification protocol is 0.3 m, the reported MPPS of viral aerosols through N 95 and N99 facepiece respirators was < 0.1 m (Eninger et al. 2008b). Small part icle size along with low inf ectious dose, high penetration through filter media, possible shielding effect of aggregation, and encasem ent effect of foreign substances are all challenges that anti-bioterrorism and public health workers need to overcome. Stability of Viral Aerosols In addition to the above properties, a key fact or in determining the spread of disease by viral aerosols is their ability to survive and maintain infectivity, i.e., the stability of viral aerosols (Cox 1995). The stability is influenced by compounds in the spray medium and environmental conditions such as temperature, relative humid ity (RH), oxygen, and pollutant (Ehrlich et al. 1964; Songer 1967; Benbough 1971; Trouwborst and de Jong 1973; Schaffer et al. 1976; Ijaz et al. 1985; Hermann et al. 2007). Benbough (1969) inve stigated the effect of various compounds including NaCl, KCl, glucose, inositol, raffi nose, glycerol, and bovine serum albumin on the survival of Semliki Forest virus (a group A arbovirus) and observed no affect of these compounds except NaCl. The removal of NaCl from the spray suspension led to better survival of viruses especially at high RH (HRH). In a following study (Benbough 1971), he observed that polyhydroxy-compounds protected arbovi rus aerosols from virucida l effects of NaCl. The protective effect of polyols was also reported in another study of influenza A viral aerosols (Schaffer et al. 1976). Similar studies were conducted for human rotavirus by using tryptose phosphate broth and fecal matter; stability of vira l aerosols at mid-range RH was observed (Ijaz PAGE 19 19 et al. 1985). In another study, the stability of coliphage T3 was reported when dextrose, spermine, spermidine phosphate, thiourea, galacturonic aci d, glucosaminic acid, and deuterium oxide were added to the spray me dium (Ehrlich et al. 1964). Among environmental conditions, RH is the mo st important when viral aerosols are generated by wet dissemination because dehyd ration is an inevitable condition (Cox 1989). Songer (1967) studied the eff ects of RH and temperature on various viral aerosols including Newcastle disease virus (NDV), infectious bov ine rhinotracheitis virus (IBR), vesicular stomatitis virus (VSV), and Escherichia coli B T3 bacteriophage. All of the virus aerosols presented poorest survival at 35% RH; NDV and VSV survived best at 10% RH, while airborne IBR and T3 phage survived best at 90% RH. Individual variation of viral aerosols was also observed in another study where vaccinia, influenza and Venezuel an equine encephalitis (VEE) viruses were found to exhibit the best stability at 20% RH while poliovirus survived well at 80% RH (Harper 1961). Indeed, the effect of RH on in fectivity of a wide rang e of viruses such as poliovirus, influenza virus, coliphage, porcine reproductive and respiratory syndrome virus (Harper 1961; Ehrlich et al. 1964; Songer 1967; Sc haffer et al. 1976; Herma nn et al. 2007) have been investigated. These authors concluded th at lipid-enveloped viruse s prefer low RH (LRH) but lipid-free viruses survive bett er at HRH. However, sensitiv ity to RH varied among virus aerosols depending on the individual ch aracteristics of viruses. The molecular structure of virus is the key parameter that determines its stab ility and sensitivity to RH, and conditions under which the nucleic acid remains intact. For ex ample, Dubovi (1971) su ccessfully extracted infectious nucleic acid of MS2 and phi X 174 from inactivated viral aerosols, and Trouwborst and de Jong (1973) observed nucleic acid separated from protein co at during inactivation of viral aerosols at various RHs. PAGE 20 20 In these studies of stability of viral aerosols, the impi ngement device was extensively used as the sampling method (Ehrlich et al. 1964; Songer 1967; Benbough 1971; Trouwborst and de Jong 1973; Ijaz et al. 1985; Herma nn et al. 2007). It collects th e entire size range of generated viral aerosols. However, Hogan et al. (2005) observed that the collection efficiency of impingement devices depended on particle size and declined to less than 10% for lower submicrometer and ultrafine viral aerosols. As particle size increased, collection efficiency increased due to increased inertia. Therefore, collection by the impingement method is strongly biased toward the bigger particle size range of viral aerosols. This limitation of impingement methods, along with the fact that viral aerosols are present in the ultrafine and submicrometer range, may lead to inaccurate understanding of the state of the viruses. How viruses are distributed in aerosol particles as a function of pa rticle size is a critical piece of information. In this context, Hogan et al. (2005) explored the di stribution of viruses for three particle sizes, 25 nm, 150 nm, and 300 nm. However, critical parame ters that affect the infectivity of viral aerosols, such as environmental conditions and composition of the fluid medium in which they are suspended, were not considered. Polymerase Chain Reaction Analysis As one of analytical tools for bioaerosols, polymerase chain reaction (PCR) is a method to detect microorganisms even from small quantities of sample by amplifying a target nucleic acid sequence of DNA, which confers advantages of sens itivity and rapidity ov er traditional culture methods (Alvarez et al. 1995). Since PCR re lies on DNA information, the PCR value is the quantity of total microbial DNA including viable and nonviable microbes without concern about viability. For microbes having only ribonucleic acid (RNA) not DNA, Reverse Transcription-PCR (RT-PCR) is widely used to detect and amplify RNA by producing DNA PAGE 21 21 complementary to the RNA, called copy DNA (c DNA) (Mackay 2004). The PCR protocol has three steps which depend on the temperature cyc ling: (1) the double stands of DNA are melted at 94-96 C yielding two single stands of DNA (denaturation); (2) the primers anneal to the single stranded DNA by making hydrogen bonds at 50-60 C (annealing); and (3) DNA polymerase synthesizes new DNA strands comp lementary to the DNA template strands by incorporating the deoxyribonucleo tide triphosphates (dNTPs) at 70 74 C (elongation). At this increased temperature, the mismatched base s of DNA will be detached due to not having hydrogen bonds enough to withstand the increased temperature. Repeated cycles of denaturation, annealing, and elongation quickly am plify the sequence of interest exponentially (Lodish et al. 2003). The amount of amplifie d product by this protocol is observed by fluorescence signal, caused by incorporating a probe. The probe containing both a reporter dye and a quencher is complementary to the target sequence. During the elongation step, polymerase cleaves the probe, releasing the reporter away fr om the quencher. Therefore, the fluorescence intensity of the reporter dye is proportional to the amount of amplification (O'Connell et al. 2006). Filtration Filtration is the most common method for aeros ol removal and has been used extensively in HVAC systems as well as in respiratory protec tion devices with the advantages of simplicity, versatility and economical collection of aerosols (H inds 1999b). The ability of filters to collect particles is described by their collection efficiency defined as the fraction of impinging particles retained in the filter, and pressure drop which is related to ener gy cost. The five basic mechanisms associated with filtration collec tion are inertial impacti on, diffusion, interception, gravitational settling, and electr ostatic attraction. Large part icles unable to quickly adjust PAGE 22 22 themselves to the changing gas streamline near the fiber will cross the streamline and hit the fiber by inertial impaction. Depending on the ratio of terminal settling velocity of particle and face flow velocity, very large particles are deposited on the fi lter by gravitation settling. In contrast, small particles encounter the fiber due to Brownian motion. When particles follow the streamline perfectly ( i.e ., they have negligible inertia, gravitational settling and Brownian motion), they are collected by interception on the filter fiber due to its finite size. Lastly, charged particles, charged fibers, or both induce electrost atic attractions ( i.e., coulombic forces and image forces) and result in particle deposition on the filter (Hinds 1999b). Aerodynamic diameter (equivalent diameter to a spherical particle wi th a unit density of 1g/cm3) is the key parameter in characterization of filtration (Hinds 1999d) ; nevertheless, it is not the only factor to be consider ed in the collection of bioaerosol s. The physical properties of microorganismsincluding shapes of aerosols and surface structureare al so important factors in collection on the filters. According to Qian et al. (1998), penetrati on by polystyrene latex spheres was higher than that of Micobacterium chelonae a rod-shaped bact erium, of similar aerodynamic size. A similar study reported that the penetration by rod-shaped organisms was lower than that by spherical organisms (Willeke et al. 1996). In another study (Jankowska et al. 2000), slightly lower collection efficiency of vari ous filter media for fungal spores than that of potassium chloride (KCl) particles of the same aerodynamic size was reported due to breakup of spore aggregates, which is different among va rious fungal spore species depending on the surface such as spiny structure. When fiber materials are used in HVAC system as well as in respir atory protection, they collect various substances in the air including du st as well as bioaerosols. The collected bioaerosols can remain viable or proliferate unde r suitable growth conditions such as sufficient PAGE 23 23 nutrient, proper humidity and temperature (Maus et al. 2001). The colonization of bacteria and fungi in the air filter used in the HVAC system was observed in previous studies (Kemp et al. 1995a; Kemp et al. 1995b; Simmons and Crow 1995). Furthermore, the survival of bacteria in various types of surgical masks a nd respirator filters was also doc umented (Brosseau L. M. et al. 1997). It has been also shown that building ma terials such as wallpaper and gypsum boards can be a source of microbial air contamination wh en the growth of microorganisms is supported by sufficient moisture and nutrients (Nielsen et al. 1998). Studies on the growth and survival of microorganisms on two different air filtration medi a reported the survival of a wide range of fungal species and bacteria on the fiberglass medi um that had significantly high water content. After 6 weeks of use, the accumulation of dust on the multi-layered polymer material provided nutrients for the growth of microorganisms (Foa rde et al. 1996). However, the growth of microorganisms can be inhibited when the growth medium dries out, suggesting better survival of microorganisms at a favorable RH (Heldal et al. 1996). Other researchers observed that microorganisms did not multiply in unused filter media at low RH (RH < 70%) but the growth of microorganisms was induced only where suffici ent moisture and nutrients were possessed (Kemp et al. 1995a). Simmons and Crow (1995) observed fungal growth at HRH ( > 70 %), which was supported by the presence of air dust and/ or cellulose fibers in the filter. Regarding respirators, rapid fungal growth in the respirator made of cellu lose was observed in the humid storage environment (Pasanen et al. 1993). When the respirator is worn for several hours, the humidity and nutrients in the resp irator may be increased due to exhalation and saliva containing various components, which can be either nutrient or antimicrobial agents (Wang et al. 1999). Microorganisms collected on filter are of great concern due to microbial contamination of ambient air by releasing byproducts or by their re-entrainment along with the adverse health PAGE 24 24 effects of bioaerosols. Various studies (Qia n et al. 1997; Willeke a nd Qian 1998; Wang et al. 1999) reported re-entrainment of the surviving mi croorganisms on the filter into the air passing through the filter media. Even though an HVAC system prevents the contamination of indoor air by microbial contaminants enteri ng from outdoors, once their growth occurs in the system, they appear in returned air at a higher level than in the outdoor air (Kowalski and Bahnfleth 1998). Similar studies also demonstrated that air condi tioning systems contribute to increased microbial concentrations in ventilated r ooms (Hugenholtz and Fuerst 1992). For respirators, a study (Qian et al. 1997) on the re-entrainment of bacteria and solid particle s from N95 respirators observed that re-entrainment of particles smaller than 1 m does not exceed 0.025 % at low RH, even at high air velocity corresponding to violent sneezing and coughing ( e.g. 300 cm/s). Meanwhile, re-entrainment of larger particles into air is sign ificant at the same re-entrainment velocities and low RH level of 22 %. The larger particles can be aggregates of bacteria or the attachment of bacteria to large inert particles. The othe r study (Jankowska et al. 2000) compared the reentrainment of biological particles to that of in ert particles from the f ilter and reported that the re-entrainment of fungal spores was higher than that of KCl particles du e to deaggregation of fungal spores. Due to re-entrainment and resistance caused by mi crobial growth, the performance of a filter can deteriorate over time Therefore, special care should be taken in handing, storage, or reuse of the filters, and fr equent inspection and maintenance should be conducted. From these two perspectives, one bei ng the prevention of contamination of ambient air by re-entrained microorganisms and the othe r being the extension of the lifetime of the filtration system by preventing proliferation of micr oorganisms in the filter, it is imperative that collected microorganisms are deactiv ated. Therefore, in recent year s, there have been efforts to PAGE 25 25 incorporate antimicrobial materials into air f ilters to destroy or inhibit the growth of microorganisms (Foarde et al. 2000; Cecchini et al. 2004). Iodine as a Disinfectant Elemental halogens (Cl2, Br2, I2, etc.) exist as diatomic molecules and form saltlike compounds with sodium and other metals (Prescott et al. 2002). Chlorine and iodine have the characteristics of antimicrobial agents. Chlori ne is the most commonly used disinfectant in water treatment among diatomic halogens due to it s relatively low cost. However, unacceptable residual levels of chlorine are a possible disadvant age of using chlorine as a water disinfectant. Iodine is used by military, in developing count ries, and in emergency or temporary use for portable water purification. It is superior to chlorine due to the greater chemical stability of the product and less reactivity with orga nic nitrogenous contaminants in water (Bruchertseifer et al. 2003). Moreover, iodine is very stable in water over a wide p H range (6) and has low solubility in water. However, continuous consumption of iodine-t reated water is not recommended due to its adverse health effect. In aqueous solution, iodine ma y exist as various species ( e.g., I-, I2, I3 -, I5 -, I6 2-, HOI, OI-, HI2O-, I2O2-, H2OI+, and IO3 -) since iodine can form compounds in all oxidation states from to +7 (Gottardi 2001). The overall reaction of iodine in water starts from hydrolysis to form hypoiodous acid (HOI) as shown in Eq. (1-1). H ypoiodous acid then disproportionates to iodate (IO3 -) as depicted in Eq. (1-2). Equation (1-3) presents the overall reaction by combining these two reactions. According to this equation, iodine molecules are si gnificant in acidic conditions. In neutral and basic solution, iodide and triiodide co-exist as shown in Eq. (1-4). At high p H (>10), HOI dissociates to hypoiodite ion (OI-) and hydrogen ion (H+) as shown in Eq. (1-5) (Bruchertseifer et al. 2003). PAGE 26 26 I2 + H2O I+ HOI + H+ (1-1) 3HOI 2I+ IO3 + 3H+ (1-2) 3I2 + 3H2O 5I+ IO3 + 6H+ (1-3) I2 + II3 (1-4) HOI H+ + OI(1-5) Among the various iodine species, iodine molecules and hypoiodous acid have disinfection capability (Chang 1958). While hypoiodous acid is the most effective form of disinfectant, molecular iodine is important in the inactivation of mi croorganisms due to its stability over a wide p H range compared to hypoiodous acid (B rion and Silverstein 1999). It is speculated that iodine molecules penetrate the ce ll wall of microorganisms and inflict structural damage on the capsid protein (Maillard 2001). Oxidation of sulfhydryl (-SH) groups or substitution onto tyrosine and histidine residues results in the disruption of normal functions of these amino acids (Carroll 1955). Brion and S ilverstein (1999) obser ved changes in the isoelectric focusing points of MS2 virions after iodine treatment from acidic p H value to more basic values, verifying that conformational change s occur in the protein of MS2 bacteriophage. The bactericidal and virucidal properties of iodi ne were observed by Hsu et al. (1965). On the other hand, iodine does not inactivate either in fectious ribonucleic aci d (RNA) or DNA (Hsu 1964). Meanwhile, study on the spor icidal effect of iodine on Bacillus metiens spores showed a decrease of germicidal activity due to incr eased iodine decomposition (Wyss and Strandskov 1945). Generally, iodine inactivation is effective in clean water, at higher p H, at higher temperature and at higher iodine dose. When using iodine as the disinfectant for such fluids as water and air, care should be exercised due to the risk of iodine vapor ingestion and concern for PAGE 27 27 hypothyroidism. Iodine vapor is intensely irritating to mucous membranes and adversely affects the upper and lower respiratory system (ACGIH 2001). The i nhalation of iodine causes coughing, burning sensations to the mucosal, trac heal, and pulmonary tissues, and tightness in the chest because it increases ai rflow resistance in th e lungs by reducing the ability of the lungs to take up oxygen. Intense exposure to iodine ma y lead to lung disease and affect the central nervous system (OEHHA 2003; ATSDR 2004) Below the threshold limit value ( i.e., 0.1 ppm) of iodine, humans can work undisturbed. Howe ver, discomfort can be encountered at 0.15.20 ppm and work is impossible at concen tration of 0.30 ppm (Cameron 2002). Iodinated Resin Filter Media Iodinated resins have been developed to pr ovide demand-on-release of iodine residuals for disinfection. Iodine can be attached to a quaternary ammonium strong base anion exchange resin in the form of triiodide (I3 -) and pentaiodide (I5 -) ions (Marchin et al. 1997). The performance of the triiodide and pentaiodide re sin was evaluated for microorganisms and 4-log inactivation of bacteria and viruses were reporte d (Fina et al. 1982; Marchin and Fina 1989). Marchin et al. (1983) reported gr eater disinfection efficacy of pe ntaiodide resin for cysts than that of triiodide. Later, the authors also observed better performan ce of pentaiodide for disinfection of Escherichia coli in both normal and microgravity (Marchin et al. 1997). Although the iodine resin typically pr oduces a residual of 0.02 2.00 mg I2/L in water passing through the filter, significantly highe r iodine residual concentration ( i.e., 9 times) in the effluent of the pentaiodide resin than that of the trii odide resin was reported s uggesting the need of a carbon filter to capture the resi dual iodine (Fina et al. 1982; Marchin an d Fina 1989). These studies indicate that the presence of pentaiodide ions on the resi n will lead not only to greater disinfection efficiency but also to an increased level of iodine vapor downstream of the iodinated PAGE 28 28 resin compared to the triiodide resi n. The iodinated resin filter, as an electret filter, is expected to possess high removal efficiency and lower pre ssure drop than conventi onal filter media. Negatively charged microorganisms attracts polari zable iodine complexes on the filter during near-contact encounters to transf er iodine molecules (Ratnesar-S humate et al. 2008). Studies on the disinfection capacity of iodina ted resin filters for the treatment of bacteria and viruses in water were conducted three decades ago a nd reported disinfecti on capacities over 99.99% (Taylor et al. 1970; Gilmour and Wicksell 1972; Hatch et al. 1980; Fina et al. 1982; Marchin et al. 1997). However, only limited studies have been conducted on the di sinfection capacity of iodinated resin filters for ai r treatment recently (Messier 2004; Heimbuch and Wander 2006; Heimbuch et al. 2007; Eninger et al. 2008a; Lee et al. 2008; Ratnesar-S humate et al. 2008; Messier 2009). In the previous study (Ratnesar-Shumate et al. 2008), the high removal efficiency of iodine-treated filter was demonstrated for vegetative cells including Escherichia coli and Micrococcus luteus The authors also proposed a near-c ontact transfer mechanism between the iodine-treated filter and microorganisms penetrati ng the filter as an inactivation mechanism, but without solid proof. To increase the reliability of the iodine-treated filte r as a protective device against airborne pathogens and biological agents, studies on mo re resistant microorganisms and microorganisms of the smallest size are needed. Furthermore, investigation of the viability of microorganisms collected on the filter is a critical step to prove the disinfection capacity of the iodine-treated filter. Both relative humidity and temperature are important environmental factors that influence the performance of iodine-treated filters because it is expected that the disinfection efficacy of an iodine-treated filter will be in creased due to the dissoci ation and dissolution of iodine at higher temperature and RH. These fact ors and conditions need to be taken into account PAGE 29 29 in the evaluation of an iodine-tre ated biocidal filter to assess its potential use as a reliable respiratory protective device. Furthermor e, a second possible source of inactivation mechanismsI2 released from the filte ralong with the proposed near-contact transfer mechanism needs to be considered. I2 released from the filter can cause inactivation in the sampling device, whereas I2 captured by microorganisms passing th rough the filter can inactivate them in their airborne state and/or continue the inactivation process after collection in the sampling device, either bound to the particle or by dissolving into the aqueous medium. Identification of inac tivation by dissolved I2 could confound the result s in earlier reports (Messier 2004; Heimbuch and Wander 2006; Heim buch et al. 2007; Eninger et al. 2008a; Lee et al. 2008; Ratnesar-Shumate et al. 2008) that used plating methods to measure viable removal efficiency, which would require an independent experimental method to quantify the relative importance of two competing inactivation mechanisms. Research Objectives Two objectives were pursued in this doctoral study to address the challenges mentioned above: (1) Performance of iodine-treated biocidal filter media as a pr otective gear against biological agents and airborne pathogens under various environmental conditions were evaluated. To achieve this objective, a filtration syst em was used to investigate the removal efficiency of filter media and vortexing experi ment was conducted to assess the viability or infectivity of biological agents collected on the filter. Furthermore, an inactivation mechanism of the iodine-treated bi ocidal filter was assessed. (2) MS2 bacteriophage aerosols in the ultrafin e and submicrometer range were characterized by investigating infectious and non-infectious virions as a function of particle size to PAGE 30 30 understand the distribution of viru ses in the airborne state. Furthermore, the effect of relative humidity and spray medium on the inf ectivity of viral aerosols was explored. Specifically, four tasks were carried out: (1) evaluate physical and viable removal efficien cy of the iodine-treat ed filter for bacterial spores and viral aerosols (2) investigate viability and in fectivity of biological agents collected on the iodine-treated biocidal filter. (3) assess the inactivation mechanism of the iodi ne-treated biocidal filter by using various reaction solutions. (4) characterize size-classified viral aerosols influenced by relative humidity and the spray medium by using plaque assa y method and PCR analysis. PAGE 31 31 CHAPTER 2 EFFICACY OF IODINE-TREATED BIOCI DAL FILTER MEDIA AGAINST BACTERIAL SPORE AEROSOLS Objective The objective of the study presented in this ch apter was to evaluate the performance of an iodine-treated biocidal filter for bacterial spores in various environmental conditions. Viable removal efficiency (VRE), pressure drop ( P ), and the viability of collected microorganisms on the iodine-treated filter were investigated and compared with those of the untreated filter. Materials and Methods Filter Media The iodine-treated filter (JT-70-20XP-10T-100) and unt reated (JT-70-20XP-100) media tested in this study as discs of 47 mm diameter and 2 mm in thickness were provided by Triosyn Corp. Triiodide, prepared from stoichiometric amounts of I2 and potassium iodide mixed with a minimum amount of water, was contacted with a quate rnary ammonium anion exchange resin to substitute th e anion with triiodide. Due to the charges on the fibers, these filters are classified as electret filters. Detail s of the preparation are available in the patent by Messier (2004). The iodine concentration in effl uent air passing through th e iodine-treated filter can be measured by the OSHA analytical method (ID -212) for iodine in workplace atmospheres. The iodine sampled in the impinger medium (1.5 m M Na2CO3 and 1.5 m M NaHCO3) can be analyzed as iodide by ion chromatography (OS HA 1994). The measured iodine concentration was 0.004 mg I2/m3. Test Microorganisms Bacillus subtilis vegetative cells were supplied by the Department of Microbiology and Cell Sciences at the University of Florida for the production of B. subtilis spores. B. subtilis is a Gram-positive, non-pathogenic, rod-shaped bacterium 2.0.0 m in length and 0.7.8 m in PAGE 32 32 width (Prescott et al. 2002). B. subtilis spores are commonly used as a surrogate for B. anthracis spores, which were the bioterrorism agent us ed in 2001 (CDC 2004). For sporulation, the African violet method (Afr ican violet soil 77.0 g, Na2CO3 0.2 g, and sterile deionized (DI) water 200.0 mL) suggested by the American Type Cult ure Collection was used (ATCC 1998). The agar was prepared by mixing nutrient agar with 25% extract of African violet soil and 75% sterile DI water. B. subtilis was inoculated in this ag ar slant and incubated at 36 oC for one week to produce spores, which are 0.8 ~ 1.2 m in length with either a s pherical or ellipsoidal shape (Ricca and Cutting 2003). After spore production, b acterial growth was harvested into 2 mL of sterile DI water and poured into a sterile glass tube. The gl ass tube containing the spore suspension was heated in a water bath at 80 oC for 30 mins to kill vegetative cells. After cooling, the spore suspension was diluted with 5 mL of sterile DI water and centrifuged at 3500 rpm (Model 225, Thermo Fisher Scientific Inc., Atla nta, GA, USA) for 5 mins. The supernatant consisting of cell debris was then removed. Th is process was repeated twice more, and the spores were resuspended in 5 mL of sterile DI wa ter. After this purifi cation process, the spore suspension was stored in a refrigerator at 4 oC before experimentation. Microscopic observation of the spore suspension after applying the ma lachite green spore-st aining technique (Munro 2000) demonstrated the purity of the culture by s howing the majority to be endospores, with only a minute amount of cell debris. Experimental System The experimental system for evaluating the rem oval efficiency is shown in Figure 2-1. A six-jet Collison nebulizer (Model CN25, BGI Inc ., Waltham, MA, USA) was used to aerosolize the spore suspension with a flow rate of 7 Lpm (liters per minute). The spore suspension in the nebulizer was made by dispersing 0.1 mL of pur ified spore suspension in 150 mL sterile DI PAGE 33 33 water. The aerosolized suspension was dried with filtered compressed air in a 2.3-L glass dilution chamber. A flow rate of 15 Lpm, whic h corresponds to a face velocity of 14.2 cm/s, was used and controlled by a calibrated rotameter. Based on the velocity, flight time through the 2 mm filter is estimated to be 14 ms. This face velocity, used by Triosyn Corp., corresponds to certification testing of 100 cm2 media (Di Ionno and Me ssier, 2004) at the 85 Lpm flow rate suggested by the National Institute for Occupa tional Safety and Health (NIOSH 2005). The concentration of bacterial spore aerosol for challenging the test filter was 1.2 104 3.2 104 colony-forming units (CFU)/m3. Pressure drop across the test filter disc was monitored using a Magnehelic gauge measuring 0 in. H2O and recorded every 20 minutes. An Andersen sixstage viable impactor (Model 10-820, Thermo El ectron Corp., Waltham, MA, USA) was used to classify generated bacterial spores and those that penetrated the test filte r. After sampling, glass Petri dishes filled with nutrient agar were re moved from the impactor, reversed, and incubated for 24 hrs for enumeration of microorganism growth. A glass fiber filter (AP 1504700, Millipore Corp., Bedford, MA, USA) was placed downstream of the impactor to capture spores not collected by the sampler, if any, to prevent contamination of ambient air. Because the cut size of the sixth stage of the impactor (0.65 m) is smaller than the nominal size of a B. subtilis spore (1 m), it is unlikely that any spores remained in the downstream air of the impactor. However, spore fragments that were not re moved by the impactor were removed by the downstream filter. The experiments were conduct ed at three environmental conditions: room temperature (23 2 oC) and low RH (35 5 %) (RT/LRH), room temperature and high RH (95 5 %) (RT/HRH), and high temperature (40 2 oC) and HRH (HT/HRH). An increased disinfection efficacy of iodine was expected at high temper ature and high RH due in part to iodines sublimation and dissolution. For the experiment s at high temperature the dilution dryer was PAGE 34 34 wrapped in an electronically controlled heating jacket. Hi gh RH was achieved by adding humid dilution air to the system. Viable Removal Efficiency The VRE of the test filter was calculated by en umerating bacterial growth in agar plates of two impactors, one downstream of the test f ilter and the other for control, which has no test filter. The VRE was determined by using Eq. (2-1). VRE (%) = 100 1 t pN N (2-1) where Nt is the total number of viable s pores collected in the control and Np is the number of viable spores collected downstream of the test filter. The entering bio aerosol concentration was measured by collecting spores at all six stages of the impactor with no test filter for the first and last 5 mins of an experimental run. The run time of 5 mins was chosen to prevent overloading of spores on the agar. The average number CFU of the two measurements was used to determine the entering bioaerosol concentration for 2 hrs of experimental run. Due to the expected low penetration of spores through the te st filter, the impactor downstream of the test filter contained only the sixth-stage agar plate. The agar plate was replaced w ith a fresh one every 20 mins for 2 hrs to avoid overloading and dehydration of the agar. Five 2-hr tria ls were conductedthe total evaluation time for each filter was 10 hrsand three filters were tested ( i.e. 15 trials). However, due to the stability of results seen at RT/LRH and tim e constraints, only two iodinetreated filters were tested for two 2-hr runs in other environmental conditions ( i.e. four trials). Agar plates containing more th an 300 colonies were counted following the positive hole method recommended by the manufacturer (T hermo Electron Corporation 2003). Viability of microorganisms on the filter PAGE 35 35 After the filtration experiment, the test filter disc was removed from the filter holder in the experimental apparatus and subjected to the vortexing experiment to determine the viability of the spores collected on the filter. The filter was immersed in 40 mL of sterile DI water in a 250 mL beaker and agitated with a vortex mi xer (Model M16715, Barnstead, Dubuque, IA). After 1 min of vortexing, 1 mL of sample was withdrawn for measuring the viability of the extracted spores in the origin al solution, and another 1 mL wa s withdrawn and measured after appropriate dilution (10-n). The same procedure was repe ated after 2, 3, 5, and 10 mins of vortexing time without changing the solution. Thus, the count of extracted spores, CE was determined by using Eq. (2-2). 2 110 V V CFU Cn E (2-2) where CFU is the number of colony-forming units counted, V1 is the volume of extraction fluid (1 mL), V2 is the volume of diluted suspension spread on the agar plate (1 mL), and n is the dilution factor. The total viab ility of the extracted spores was calculated by averaging the number of viable spores at all vortexing times. To compare the results of the iodine-treated and untreated filters, we defined surv ival fraction as the ratio of the extracted spores in the vortexing solution to the spores collected on the test filter ( CE/CC). CC is determined by the total count in the control impactor multiplied by the VRE of the filter. In aqueous so lution, the resin surfaces may release iodine molecules that also may deactiv ate spores. This reacti on raises concerns that spores can be deactivated in the vortexing solution by free residual iodine rather than deactivation solely on the filter. To investigate this possibil ity, the solution after vortexing a clean iodine-treated fi lter at each designated time was inoc ulated with a spore suspension of known concentration. After 10 mins of exposure time, spore concentration was measured to determine the free residual iodine effects. The concentration of iodine in the vortexing solution PAGE 36 36 was also examined by the DPD ( N N -diethylp -phenylenediamine) colori metric method adapted from Standard Methods for the Exami nation of Water and Wastewater 4500-CI (APHA 1995). Ten mL of solution vortexed with the iodine-treated filter wa s analyzed at 530 nm by using a DR/4000 V Spectrophotometer (Hach, Loveland, CO, USA). Iodine in the solution reacts with DPD forming a pink color, the intensity of which is proportional to the tota l iodine concentration (Hach 2003). The effect of vortexi ng alone on the viability of s pores was also investigated by following the same vortexing procedure with a spore suspension of known concentration. Results Removal Efficiency Figure 2-2 shows the size dist ribution of the ente ring spores collected by the impactor. As shown, the majority of the en tering spores were in the 0.65 ~ 2.1m range, indicating they were predominantly singlets. As shown in Tabl e 2-1, both iodine-treated and untreated filters displayed a high VRE (> 99.996 %) at RT/LRH due to the high mechanical removal efficiency. Differences in the VRE should be distinguishable at a much higher upstream concentration, but this would overload the impactor and filter in our experimental conf iguration. In other environmental conditions ( i.e. RT/HRH and HT/HRH), the iodi ne-treated filter also achieved high VRE ( 99.998 %). It should be noted that even when the filter did not show complete removal, in most cases only one or two CFU pe netration was detected downstream. There was no difference in any 2-hr interval, indicating that the performance did not deteriorate over time during the 10-hr or 4-hr expe rimental runs. Raw data ar e presented in Appendix A. Pressure Drop Since pressure drop is an important parameter in practical applications, P was recorded every 20 minutes. Under the operating condition, the initial pressure drop was approximately PAGE 37 37 423 Pa (at 14.2 cm/s) and was maintained thr oughout the entire expe riment with almost negligible variation. There was no observable di fference in pressure drop between the iodinetreated and untreated filters. Survival Fraction To determine the viability of the collected spores, both iodine -treated and untreated filters were vortexed to extract spores from the filters. A larger number of s pores extracted from the untreated filter was enumerated than from the iodine-treated filter at RT/LRH. No increase of extracted spores from the test filters was obs erved as the vortexing time increased. Although both survival fractions were low, the survival fraction of the iodine-treated filter was significantly lower than that of the un treated filter, which was confirmed by t -test ( p -value < 0.05). At RT/HRH and HT/HRH, the survival fraction of the i odine-treated filter showed around one log unit higher value than that at RT/L RH. This higher surviv al fraction can possibly be explained by the loss of iodine from the filter due to increased sublimation of iodine at HT and dissolution through the hydrolysis of iodine at HRH. To test this hypothesis, we measured the iodine concentration in the vortexing solution of the iodine-treated filter by the DPD colorimetric method. The values (mg I2/L) at RT/HRH (0.40 0.03) and HT/HRH (0.30 0.03) were lower than that at RT/LRH (0.90 0.03). Statistical significan ce between RT/LRH and the others was observed by performing one-way ANOVA ( p -value < 0.05). Meanwhile, the difference between RT/HRH and HT/HRH was not significant ( p -value > 0.05). We note that n is small ( i.e. 2) and measurement uncertainty of surviv al fractions is large at both RT/HRH and HT/HRH. To investigate the effect of residual free iodine in the vortexing solution on the survival fraction of the extracted spores, spores were inoc ulated into the solution after vortexing a clean PAGE 38 38 iodine-treated filter at each designated vortexing ti me. As shown in Figure 2-3, the effect of the extracted iodine did not increase as vortexing time increased. The average ( S.D) fraction of spores was 0.856 ( 0.014) and 1.01 ( 0.03) in the iodine-treated and untreated solution, respectively, indicating that the iodine extracted from the iodine-treated filter during vortexing decreased the viability of spores in the solution by ~15%. Acco rdingly, the survival fraction of spores on the iodine-treated filt er was corrected by this amount. The effect of vortexing alone on the viability of spores was also examined. A spore suspension of known concentration was vortexe d for each designated time, after which the viability of each was examined. The relative fraction obtained by dividing the number of viable spores after each vortexing time w ith that at zero vortexing time was calculated. The average ( S.D) fraction was 1.03 ( 0.15), demonstrating that 10 mins of vortexing had a negligible effect on the viability of spores. The corrected survival fraction consid ering only the effect of free residual iodine is presented in Table 2-2. After vortexing, a tested and an unused iodi ne-treated filter were examined under a scanning electron microscope (SEM) (FESEM6335F, JEOL, Japan) to look for spores not extracted from the filter. As shown in Figure 2-4, a few micron-sized particles remained in the tested iodine-treated filter, whereas th e unused filter was free of particles. Discussion In practical applications, a desirable filte r medium will provide high aerosol removal efficiency with an acceptable P depending on the applications. Fo r a ventilation system, a large P imposes high energy and maintenance costs. In respiratory protection, a large P translates into breathing exertion with a respirator, whic h may impair the agility or compromise the mobility and endurance of personnel in the battlefiel d or workers in disaster zones. The test PAGE 39 39 filter medium can be applicable to both ventila tion systems and respiratory protection devices. Since the pressure drop of the f ilter is directly proportional to f ace velocity with the assumption of laminar flow inside the filter (Hinds 1999b), the expected pressure drop of the test filter at a face velocity of 5.3 cm/s is around 157 Pa, which is much less than the military standard of 392 Pa for HEPA filter media (U.S Army 1998). In the case of respirator application, the pressure drop of the test filters can be calcu lated for the face velocity of 7.8 cm/s, which is achieved when the flow rate of 85 Lpm for the NI OSH respirator certification testing is applied to commercially available facepiece respirator s (Barrett and Rousseau 1998). The calculated pressure drop of the test filters is 224 Pa, whic h is much less than the inhalation resistance of 343 Pa permitted by NIOSH for certified respirators (N IOSH 1995). Incorporation of iodine on the resin filter media did not affect P The test filter media exhibited high VRE (> 99.996%) for bacterial spores. This value is as high as th e filtration efficiency of NIOSH-approved N95 and P100 respirator filters for B.globigii spores (99.87 % and 99.98 %, respectively) using 85 Lpm flow rate as reported in a recent study (Richardson et al. 2006). There are great concerns about the growth of microorganism s collected on the filter, which may result in the release of byproducts and re-entrainment. It also poses a hazard to workers who handle the disposal of a microorgani sm-loaded filter. It has been shown that fibrous building materialsincludi ng insulation substances and cei ling tileserve as nutrients for the growth of microorganisms under sufficient relative humidity (Ezeonu et al. 1994; Chang et al. 1995). Research about the e ffect of air filter media on the vi ability of bacteria showed that fiber materials did not have an in hibitory effect on the survival of microorganisms even if they do not grow (Maus et al. 1997). Sensitive cells lose their viability in less than three days after collection, but resistan t bacteria such as B. subtilis spores can retain viability on the filter for a PAGE 40 40 much longer time (Wang et al. 1999). As prev iously mentioned, the complex structure of bacterial spores protects cellu lar components by developing antim icrobial resistance, and low concentrations of chemical germinants can cause the spores to germinate (Moir et al. 2002). A study on the killing mechanism of spores by chem icals used in decontamination procedures demonstrated that spores can germinate even afte r they are treated with nitrous acid, while those treated with Betadine containing 1% available iodine do not germ inate (Tennen et al. 2000). Our study demonstrates such a benefit of incorp orating iodine with filtration for biocidal applications. Surviving microorganisms on filters can re-ent rain into the air pa ssing through the filter medium, which has been reported in several studies (Qian et al. 1998; Wang et al. 1999; Rengasamy et al. 2004). A study on the reaerosoliza tion of bacteria and so lid particles from N95 respirators observed that the reaerosolization of particles smaller than 1 m is insignificant (< 0.025%). Reaerosolization of larger aggregates of bacteria or bacteria attached to large inert particles, however, is significant at the same reaerosolization velocities, which correlate with violent sneezing and coughing, and at low (22%) RH (Qian et al. 1997). The reentrainment of fungal spores was higher than that of KCl par ticles due to disaggregat ion of fungal spores. Moreover, the rate is different among vari ous fungal spore species depending on the surface structure (Jankowska et al. 2000). The pres ent study showed very low extraction (6.9 10-4 1.6 10-4) by vortexing. This value is much lowe r than that reported in a prior study for B. subtilis from polycarbonate filters, where the vortexi ng method exhibited extr action efficiency of 85% (Wang et al. 2001). In other words, the spores were securely trapped in the filter matrix of our test filters, resulting in inefficient extraction. This phenomenon is supported by the SEM images shown in Figure 2-4. The bacterial spores are attached to the fi brous surface due to van PAGE 41 41 der Waals forces. For an electret filter, the electrostatic interaction between the positively charged resin surface and negatively charged microorganisms further enhances the attachment. These two forces are weakened when water is presen t. Therefore, detachment is expected to be faster in water than in air. However, even w ith vortexing to enhance the dislodging, the spores were still trapped securely, implyi ng that reaerosolization from such an electret filter in air will be low. From a practical application pe rspective, the resin filter mate rial without iodine treatment is an effective medium to trap the relativ ely large bacterial spores with negligible reaerosolization. In both iodine-treated and untreat ed filters, negatively ch arged bacterial spores are influenced by attractive force with the pos itive resin surface and repulsive force due to negatively charged functional groups on the filter medium. Specificall y, the resin surface and the iodide ions remaining after depletion of iodine molecules from triiodide have similar attractive and repulsive force as the untreated filt er. Therefore, both iodi ne-treated and untreated filters presumably have similar retention of the b acterial spores, suggesting that the filter medium that is depleted of iodine over time can still serve as an effect ive medium trapping the spores. It should be noted that the efficacy of a bioc idal filter is observed for bacterial spores collected on the filter, wh ich are exposed to iodine disinfectant in the filter for several hours. The separate question about inactivation of penetrating bacteria l spores by interactions during the short penetration time ( i.e. 14 ms) is not addressed by this experiment because the number of penetrated bacterial spores is too low to distinguish a biocidal effect. Further evaluation of smaller microorganisms, which exhibit higher penetration, is warrant ed to generalize the assessment of a biocidal agent on the penetrat ing microorganisms and a pplication to a wide range of biological agents. PAGE 42 42 One important thing considered in the use of the biocidal filter is the health effect of the incorporated antimicrobial agent. Since iodine vapor irritates mucous membranes and adversely affects the upper and lower respiratory system, its inhalation can cause coughing and tightness in the chest (Cameron 2002). The iodine concentra tion in the air passing through the iodine-treated filter is as low as the detection limit of the analytical method, which is 0.004 mg/m3 (OSHA 1994). It is much less than th e 8-hr Time Weighted Averag e-Threshold Limit Value (TWATLV) of 1 mg/m3, which is the level below which a worker is expected to have no adverse health effect resulting from chronic exposure (OSHA 2000). In conclusion, both the iodine-treated and untreated filter media present effective approaches to the removal of bacterial spore aerosols. They achieve high viable removal efficiency without increasing pres sure drop by incorporating iodine as a disinfectant into the filter medium. Furthermore, the deactivation of the collected bacterial spore aerosols is enhanced by the iodine-treated filter compared to the untreated filter before the filter medium loses significant amount of iodine du e to sublimation and dissolution. PAGE 43 43 Table 2-1. Removal efficiency of the iodine-treated and untreat ed filters for bacterial spore aerosols at various environmental conditions Environmental conditions Test Filters Trial No. Challenge (CFU) Penetration (CFU) Removal efficiency (%) 1,5,7,9,10, 11,13,14,15 4.9 104 9.8 104No > 99.9980 2 9.5 1041 99.9989 3 1.1 1052 99.9981 4 8.7 1041 99.9988 6 8.0 1041 99.9988 8 6.5 1041 99.9985 Iodine-treated filter 12 5.8 1041 99.9983 1,2,4,6,7,9, 11,12,14 4.2 104 8.7 104No > 99.9976 3 6.4 1041 99.9984 5 6.7 1041 99.9985 8 6.3 1042 99.9968 10 5.6 1042 99.9965 13 5.9 1041 99.9983 Room Temp. Low RH Untreated filter 15 6.1 1041 99.9984 1,2,3 7.3 104 8.1 104No > 99.9986 Room Temp. High RH Iodine-treated filter 4 8.0 1041 99.9987 1,3,4 8.7 104 9.3 104No > 99.9989 High Temp. High RH Iodine-treated filter 2 9.0 1041 99.9989 PAGE 44 44 Table 2-2. Survival fraction of bacterial spores on both filters at various environmental conditions Environmental Conditions Test Filters Average S.D Iodine-treated filter 6.9 10-4 1.6 10-4 Room Temp. Low RH* Untreated filter 2.5 10-3 1.4 10-3 Room Temp. High RH Iodine-treated filter 5.1 10-3 5.5 10-3 High Temp. High RH Iodine-treated filter 8.3 10-3 5.8 10-3 Significant difference between the result of iodine-treated filter and untreated filter Figure 2-1. Experimental setup for bacterial aerosol system PAGE 45 45 Figure 2-2. Particle size distri bution of entering bioaerosols Figure 2-3. Relative fraction of spores in the vortexing solution of the clean iodine-treated and untreated filters PAGE 46 46 A B Figure 2-4. SEM images of the filters at 100X. A) Unused iodine-treat ed filter. B) Iodinetreated filter after vortexing experiment PAGE 47 47 CHAPTER 3 ASSESSMENT OF IODINE-TREATED FILTER MEDIA FOR REMOVAL AND INACTIVATION OF MS2 BACTERIOPHAGE AEROSOLS Objective The objective of the study in this chapter was to evaluate an iodine-treated filter medium for removal and inactivation of viral aerosols under various environmental conditions and explore inactivation mechanisms of the filter. Physical removal efficiency (PRE), viable removal efficiency (VRE), pressure drop, I2 concentration in the impinger medium, and the infectivity of viruses collected on the iodine-treat ed filter were investigated and compared with those of an untreated filter. The inactivation mechanism proposed earlier for the iodine-treated filter was examined by measuring VRE downstream of the filter using various collection media that were inert, moderately react ive and aggressively reactive to I2. Furthermore, a second possible source of inactivation mechanisms was consideredI2 released from the filter and transported to the impinger where the in activation was hypothesized to occur. Materials and Methods Test Filters Samples of the iodine-treated (polyestercotton, 125 g/m2 triiodide resin, Safe Life Corp., San Diego, CA, USA) and untreat ed (polyestercott on, Safe Life Corp., San Diego, CA, USA) filter media, both as flat sheets 1 mm thick, were tested as discs of 47 mm diameter. The information on the preparation of an iodine-treated filter is described in Chapter 2. The I2 concentration was measur ed to be 0.004 mg I2/m3. Test Microorganisms MS2 bacteriophage (ATCC 15597-B1) was selected as a re presentative virus aerosol. In the selection of a model viru s, its resistance to antimicrobial agents should be considered because resistance varies from one virus to anot her (Berg et al. 1964; Sobsey et al. 1990). MS2 PAGE 48 48 is a non-enveloped, icosahedron-shaped, single-st randed RNA with a single-capsid size of 27.5 nm, and it infects only male Escherichia coli (Prescott et al. 2002). MS2 has been used as a surrogate for small RNA enteroviruses pathogeni c to humans because they both have no lipid component surrounding the protein co at and are considered to ha ve similar resistance (AranhaCreado and Brandwein 1999; Bri on and Silverstein 1999). Freeze-dried MS2 was suspended with filtered deionized (DI) water to a concentration of 1089 plaque forming units (PFU ) /mL as the virus stock suspension and stored at 4 C. Experimental System and Conditions The experimental set-up for testing the removal efficiency of filters is shown in Figure 31. Seven Lpm (liters per minute) of dry, f iltered compressed air was passed though a six-jet Collison nebulizer (Model CN25, BGI Inc., Wa ltham, MA, USA) to aerosolize the viral suspension. The virus concentration in the Collison nebulizer was 1056 PFU/mL and was prepared by diluting 0.10 or 0.20 mL of virus stock suspension in 50 mL of sterile DI water. The aerosolized particles were dried with filtered compressed air in a 2.3-L glass dilution dryer. A flow rate of 8 Lpm, which corresponds to a face velocity of 14.2 cm/s, was used for each stream ( i.e ., control and experimental) and controlled by a calibrated rotameter. Based on the velocity, flight time through the 1-mm filter is estimated to be 0.007 seconds. This face velocity, used by Safe Life Corp., corresponds to certification te sting of 100 cm2 media (Di Ionno and Messier 2004) against the 85 Lpm flow rate specified by the National Institute for Occupational Safety and Health (NIOSH 2005). Pressure drop acros s each filter was monito red with a Magnehelic gauge measuring 0 Pa and recorded every 30 minutes. The viral aerosols entering and penetrating the test and control filters were collected in an AGI-30 impinger (Ace Glass Inc., Vineland, NJ, USA) containing 20 mL of sterile phosphate buffered saline (PBS). The collection PAGE 49 49 medium in each impinger was replaced by fresh solution every 30 mins and assayed to determine the virus concentration by using suitabl e dilution to an adequate count ( i.e ., 30 PFU). The procedures for preparing plaque assay medium ar e presented in Appendix B. Five 2-hr trials were conducted, and thus total evaluation time was 10 hrs. Since I2 and HOI are disinfective forms, an increas ed VRE of the iodine-treated biocidal filter at high temperature and increased relativ e humidity (RH) was hypothesized due possibly to iodines sublimation and to increased dissolution through th e hydrolysis of I2 to HOI. Therefore, various environmental conditions were considered: room temperature (23 2 C) and low relative humidity (35 5%, RT/LRH); high temperature (30 2 C) and LRH (HT/LRH); RT and medium RH (50 5%, RT/MRH). Because the maximum in activation of MS2 aerosolized from 0.1 M NaCl was reported to occur at 75% (Trouwborst and de Jong 1973), RHs below this level were considered. Temperature and RH were adjusted by wrapping the dilution dryer with a heating jacket and adding dry or humid dilution air to the system. Removal Efficiency Removal efficiency of viral ae rosols by the test filters can be expressed both as PRE and as VRE. The particle size di stribution (PSD) of th e aerosols entering and penetrating the test filters was measured by using a Scanning Mobility Particle Sizer (SMPS; Model 3936, TSI Inc., Shoreview, MN, USA) and the PRE was determined by using Eq. (3-1). PRE (%) = 100 1 E PN N (3-1) where NE is the number of particles entering the filter and NP is the number of particles penetrating the filter. PAGE 50 50 The VRE depends on the infectivity of viruse s collected in the impingers. The VRE was determined by counting plaques on each Petri dish from both control and experimental impingers, and calculated according to Eq. (3-1). In calculating the viral concentration, a dilution factor was used, which depends on the nu mber of transfers of the impinger solution. Thus, the viral concentration in the impinger, Cv ( PFU /mL), was determined by using Eq. (3-2). Cv = V PFUn10 (3-2) where PFU is the number of plaque-forming units, V is the volume of diluted solution, and n is the dilution factor. The final mean viral concentr ation was determined by averaging all values in each dilution. Inactivation Mechanism of the Io dine-Treated Biocidal Filter Two possible inactivation mechanisms of the iodine-treated filter were considered: (1) inactivation of viruses downstream of the filter by reaction with I2 released from the filter and (2) direct transfer of I2 during near contact as viral aerosols pass through the iodine-treated filter. Sublimation and Dissolution of Iodine Molecules Released from the Filter To investigate the effects of iodine released from the iodine-treated filter, filtered clean air passing the test filter at various envir onmental conditions was drawn into impingers containing a viral suspension of known concentra tion. The virus in the experimental impinger might lose its infectivity due both to the operation of the impinger ( e.g ., swirling effect and reaerosolization) and to the action of I2. Meanwhile, the infectivity of viruses in the control impinger will be affected only by the operation of the impinger. Therefore, by comparing the results of the control and the experimental impingers, the loss of virus infectivity by the operation of the impinger was excluded. PAGE 51 51 How I2 disinfects virus in the impinger was stud ied by using sodium thiosulfate solution to quench the r eactivity of I2 available in the impinger. The same experimental procedure described previously for sub limation and dissolution of I2 was followed except that the impinger medium was replaced by a 0.1 M solution of sodium thiosulfate. Thiosulfate anion (S2O3 2-) reacts stoichiometrically with I2 and reduces it to iodide, which is not virucidal (Ber g et al. 1964). Transfer of I2 to Viral Aerosols To investigate the inactivation mech anism of direct transfer of I2 to viral aerosols, the effect of sublimation and dissolution of I2 released from the iodine-treated filter should be excluded. The use of thiosulfate solution has a li mitation in this exclusion because it can react both with I2 existing free in the impi nger solution and with I2 residing on the MS2. Therefore, a halogen-demanding substancebovine serum album in (BSA)was used, which consumes free I2 in the impinger solution but competes less aggressively than thiosulfate for I2 on the MS2. The capacity of BSA to consume all of the I2 released from the filter was predetermined by using the same experimental configuration fo r sublimation and dissolution of I2 except that the impinger contained 0.3%, 3% and 6% BSA and a virus su spension of known concentration. The filtration experiment was then performed using the selected concentration. Viral aerosols were delivered as challenges to the iodine-treated filter and coll ected in both control and experimental impingers for 1, 5, 10, and 15 mins. The MS2 in the e xperimental impinger was compared to the penetrating MS2. For comparison, the same experi ment was performed with thiosulfate solution as the collection medium of the impinger for 15 mins. Infectivity of Viru ses on the Filter After 10 hrs of removal efficiency experiments, the test filters were retrieved from the filter holder in the experimental system and subjected to a vortex mixer (Model M16715, PAGE 52 52 Barnstead, Dubuque, IA, USA) to investigate the in fectivity of viruses co llected on the filter. The filter was immersed in 40 mL of sterile DI water in a 250-mL beaker and vortexed for a designated time ( i.e ., 0, 1, 3 and 5 min) to investigate the optimal extraction time. The vortexing solution was assayed to determine the infectiv ity of viruses and th e number of viruses ( Nv) was determined by using Eq. (3-3). 2 110 V V PFU Nn v (3-3) where V1 is the volume of extraction fluid and V2 is the volume of origin al or diluted suspension assayed with host cells. The total infectivity of extracted viruses was cal culated by averaging the results at all vortexing times because the num ber of extracted viru ses at each designated vortexing time was found to be similar. The extr acted fractionthe ratio of the infectivity count in the extraction solution to the total viruses co llected on the filterwas used to compare the result of the iodine-treated f ilter with the untreated filter. Effects of Free Iodine Molecules In an aqueous suspension for the vortexing expe riment, the resin surfaces are expected to release I2 that can inactivate viruses. This reaction raises a question whether viruses lose their infectivity in the extract solution due to the free I2 residual or on the filter. To investigate this question, the solution after vortexi ng a clean iodine-treated filter for a designated length of time (0, 1, 3 and 5 min) was inoculated with a viru s suspension of known concentration. Because it took 15 minutes to finish the vortexing experiment including dilution and a ssay, the infectivity of virus in the mixed suspension was analyzed after 15 minutes of exposure to the free I2 in the suspension. The I2 concentration in the vortexing so lution was analyzed by the DPD ( N N diethylp -phenylenediamine) colorimetric method adapted from Standard Methods for the Examination of Water and Wastewater 4500-CI G (APHA 1995). Ten mL of solution vortexed PAGE 53 53 with the iodine-treated filter was analyzed at 530 nm by using a DR/4000 V Spectrophotometer (Hach, Loveland, CO, USA). I 2 in the solution reacts with DP D to form a pink color, the intensity of which is proportional to the total I2 concentration (Hach 2003). The effect of vortexing alone on the infectivity of viruses wa s also investigated by following the same vortexing procedure with a virus suspension of known concentration. Results Physical Removal Efficiency and Pressure Drop The PRE of the test filters was determin ed by comparing the PSDs of the aerosols entering and penetrating the test filters as shown in Figure 3-2. The PSD of the aerosols entering the test filters showed its mode at approximately 25 nm. As a ba seline, sterile DI water without virus was aerosolized from the nebulizer and the PSD of that was measured, defining the background noise. Therefore, the PSD of the aeros ols above the noise level in the window from 9.82 to 162.5 nm was considered for the calculation. The PRE (mean SD) of the iodine-treated and untreated filters for this size range were 41 3 % and 39 2 %, respectively. Statistical evaluation of the two values by a one-tailed students t -test indicated that the difference was insignificant ( p -value > 0.05). The initial pressure drop of the test fi lters was around 50 Pa and the variation in pressure drop during the entire experiment was negligible. This value is much less than the inhalation and exhalation resistan ces of the respirator certified by NIOSH, which cannot exceed 343 Pa and 196 Pa, respectively (CFR 2002). No significant difference in the pressure drop between the iodine-treated and untreated filters was observed. Viable Removal Efficiency PAGE 54 54 The VRE of the test filters was calculated by an alyzing the infectivity of viruses collected on both control and experimental impingers for challenging and penetrating viruses from the filter. The result is presented as an average of five 2-hr experimental runs for each filter indicated as No.1 and No. 2 in Ta ble 3-1 (raw data are available in Appendix C). As shown, the iodine-treated filter presented a significantly higher VRE than that of the untreated filter ( p -value < 0.05) at various environmental conditions. At HT/LRH, a significantly higher value of the iodine-treated filter than th at of the other conditions was observed, according to one-way ANOVA ( p -value < 0.05), due to increased release of I2 from the filter. Meanwhile, the difference between RT/LRH and RT/MRH was not significant ( p -value > 0.05), indicating that the release of HOI into air due to the hydrolysis of I2 at increased RH is negligible. Inactivation Mechanisms of the Io dine-Treated Biocidal Filter The effect of sublimation and dissolution of I2 was investigated by using the impingers containing a virus suspension of known concentrati on either in the PBS or sodium thiosulfate solution. As shown in Table 3-2, no surviving vi rus was detected in the experimental impinger until > 104 PFU in the PBS was added to the impinge rs. As the virus concentration in the impinger increased, the number of surviving viru ses also increased. Meanwhile, the survival fraction of viruses in the thiosulfate solution was mu ch higher than that in the PBS. Most viruses suspended in the thiosulfate solution survived in the experimental impinger due to quenching by reaction with thiosulfate of the I2 released from the iodine -treated filter and/or I2 transferred to viral aerosols. Hatch et al. (1980) proposed spontaneous dissociation of I2 from the polyiodide resin complex as one of three possible in activation mechanisms of their iodinated resin filter in water treatment. In anothe r study (Marchin et al 1983), acquisition of I2 by a cyst during passage through an iodinated resin column was hypothesized. The authors observed that PAGE 55 55 cysts regained viability due to reduction of I2 by thiosulfate solution for up to 3 mins. A more recent study (Brion and Silverstein 1999) reporte d reversal of MS2 in activation after a few minutes (< 5 mins) of iodine treatment by adding 0.3 % BSA. It must be noted that these studies were performed in water, so their applicability to inactivation mechanisms of iodine in air treatment is uncertain. In the experiments measuring I2 demand of BSA, various co ncentrations of BSA were evaluated. As shown in Table 3-2, the survival fractions of MS2 in the experimental impinger having 3 % and 6 % BSA were similar to thos e in the control impinger (~0.95). The result indicates that both 3 % and 6 % BSA solutions contain sufficient protein to exhaust I2 released from the filter and thus isolate MS2 in the experimental impinger from inactivation by I2 in solution. The history of iodination of albumin s suggests significant dependence on conditions. Muus et al. (1941) reported rapid uptake of 15 wt% iodine by horse serum albumin (HSA) from ~0.2 N I2/KI in aqueous ethanol and Shahkrokh (1943) a dded 8 wt% iodine to HSA with a similar concentration of I2/KI in water. Hughes and Straessle ( 1950) incorporated 30 molar equivalents of iodine into human serum albumin in 0.1 N aqueous I2, converting 70% of L-tyrosine residues into diiodotyrosine. Small-scale preparations ad ding chloramine-T to similar concentrations of K131I in water achieved fast and efficient incorporation of the small amount of 131I into human growth hormone (Greenwood et al. 1963), BSA (Opresko et al. 1980) and BSA microspheres (Smith et al. 1984). Lee and Ellis (1991) proposed the reaction with iodine solutions as a method to visualize serum albumins on polyacrylamide ge ls. However, Shahkrokh (1943) also showed that the extent of reaction of HSA with I2 falls off rapidly with d ecreasing concentration and Portenier et al. ( 2001) reporte d that an equimolar amount of BSA did not suppress the bactericidal activity of a 0.2% solution of I2/KI. PAGE 56 56 In the experiments in which aerosolized MS2 pe netrated the iodinated filter, collection in a medium containing thiosulfate effectively neut ralized all of the iodine released, whether displaced and captured or dissociated, as no decr ease in viable penetration was observed (shown in Figure 3-3). In contrast, a similar experime nt in which the penetrating particles and free iodine were collected in 3% BSA medium, showed that half the penetrating MS2 virions were inactivated initially and a modera te increase in survival was seen after 10 minutes. The initial observation is consistent with the mechanis m proposed by RatnesarShumate et al. (2008) because the data in Table 3-2 show that the captu re medium is able to consume all of the free iodine coming off the filter. Thus, at least half of the MS2 viral particles penetrating the filter in this experiment appear to have acquired and bound a lethal dose of I2 as they traversed the iodine-treated filter. The distinguishable increas e of surviving MS2 at 10 mins of collection time parallels a delayed reactivation of MS2 obser ved in aqueous iodine solutions (Brion and Silverstein 1999), and it is tempting to conclude that the deactivation processes in water and in this system are similar after iodine has been transported to the virion. However, some combination of direct transfer of I2 from the filter plus dissociation of I2 from weaker binding sites on penetrating particles repr oduces their general conditions a nd appears to cause almost half the observed inactivation of viral aerosols penetra ting the iodine-treated f ilter and collected in PBS medium. After submission of a manuscript describing this effect, Triosyn Corp. (Messier 2009) disclosed data showing a thres hold for inactivation of MS2 and of Staphylococcus aureus at 0.5~0.6 ppm I2 in PBS medium, which is consistent with results presented he rein and defines a boundary condition to anticipate sign ificant interference by dissolved iodine. We then verified that the data reported by Heimbuch and Wander (2006) and by Heimbuch et al. (2007) were measured under conditions th at inactivation by free I2 did not contribute significantly. PAGE 57 57 Eninger et al. ( 2008a) collected MS2 aerosol s penetrating an iodinated medium onto gelatin-coated plates, which they washed out in to water and plated in a plaque assay. They observed no kill of MS2 and concluded that the treatment was ineffective. However, their observation of no inactivation by i odine during the steps of their workup that were executed in water shows clearly that the overw helming excess of protein in th eir collection surface consumed all of the iodine displaced, re leased or captured from the iodi nated medium. Whereas their experiment thus does not support the conclusion that the treatment is inactive, in the absence of measurements of I2 concentrations in the impingers we can make no quantitative statement about the relative importance in our data set of these potentially competing pro cesses for inactivation. However, we note that, even though sufficient I2 is released to confound the environment in the impingers, the airborne concentration of I2 released from the filter was much less than the 8-hr Time Weighted Average-Threshold Limit Value (TWA-TLV) of 1 mg/m3, the level below which a worker is expected to have no adverse health effect resulting from chronic exposure (OSHA 2000). Hence, whatever activity is present is realistically available for use in respiratory protection. Effects of Free Iodine Molecules and Extracted Fraction To account for the effect of free iodine in th e extract solution, the infectivity of viruses mixed with the vortexing solution from a clean iodine-treated filter after each designated vortexing time was analyzed and ex pressed as survival fraction ( CS/CI, CS: surviving MS2, CI: initial MS2 in the suspension). The average valu e of the survival fracti on at all vortexing times, 0.17 ( i.e ., 83% attenuation), was used to correct the value for the inf ectivity of viruses collected on the filter. As presented in Table 3-3, the I2 concentration in the vortexing solution measured by the DPD colorimetric method was around 1.0 mg/L I2. Some I2 was released from the iodine- PAGE 58 58 treated filter before the start of the vortexing procedure, designated as vortexing time. No further increase of I2 extraction from the filter by in creasing vortexing time was observed. The infectivity of viruses collected on the f ilter is presented as the extracted fraction ( CE/CC, CE: MS2 extracted from the filter, CC: MS2 collected on the filter). Cc for the iodinetreated filter was determined from the VRE of the untreated filter becau se both iodine-treated and untreated filters had a similar PRE. The effect of vortexing on the viruses was negligible because the infectivity of viruses vortexed at va rious times did not have observable variation. Table 3-4 presents both observed and corrected values of the ex tracted fraction. The corrected values were determined by dividing the observe d values by the surviv al fraction (0.17) to consider the effects of free I2. As shown, no significant difference in the corrected extracted fraction between iodine-treated and untreated filters at the sa me environmental condition was exhibited ( p -value > 0.05). Both iodine-treated and untreated filters tested at MRH showed the lowest value among the survival fractions presumab ly due to the sensitiv ity of MS2 to the MRH (Dubovi and Akers 1970). The lower values of free I2 from the iodine-treat ed filter tested at HT/LRH and RT/MRH than that at RT /LRH indicate measurable loss of I2 from the iodinetreated filter. Although the filter constantly experienced loss of I2, it was observed that the efficacy of the iodine-treated biocidal filter did not deteriorate during 10 hrs of experiment. After vortexing, one tested filte r and one unused iodine-treated filter were examined under a scanning electron microscope (JSM-6330F, JEOL Ltd., Tokyo, Japan). As shown in Figure 34, abundant particles were observed in the test ed filter compared to the unused filter. Discussion Intrinsic differences in test methods co mplicate comparison of PRE and VRE values measured for test filters. The PRE was measured for ultrafine particles ( i.e. 9.82 to 162.5 nm), whereas the VRE was measured over the entire pa rticle size range generated from the nebulizer. PAGE 59 59 Even if the PRE for the entire pa rticle size range is ca lculated by particle c ounting, its value will still be different from the VRE because of a ggregation of virus aerosols and fewer counts of viable virus available for disaggr egation in smaller particles than in bigger particles. A viral aggregate is measured by the par ticle counter as one particle, but it can be assayed as several viruses after collection in the impinger because of dispersion in the collection medium. The number of viable viruses in a big particle is larger than that in an ultrafine particle; thus, the contribution of larger particles collected in the im pingers to the infectivity results will be much greater than that of ultrafine pa rticles. This effect was obser ved in a prior study (Hogan et al. 2005), which reported that the probability of contai ning viable viruses increases with the size of particles from MS2 suspension In the experiment for sublimation and dissolution of I2, the observed increase of survived viruses as virus concentration in the impinger increased is presumably due primarily to exhausting the supply of I2 but might also include some shielding effect of aggregated/encased viruses if the aggregate persists in the impinger. Berg et al. ( 1964) reported that deactivation of viruses by iodine follows first-order reaction kine tics, and thus reaction rates of iodine with viruses depend on the number and availability of vital sites on the vi rion. They mentioned a lagged deactivation curve of iodine due to viru s clumping and the necessity of time for virus clumping to be separated. A study of the surv ival of viral particle s in aqueous suspension irradiated with ultraviolet light demonstrated th at virus survival was st rongly dependent on the degree of aggregation among the viral particles (Galasso and Sharp 1965). The SEM images of the tested filter in Figure 3 4 show that many particles still remained on the filter after extrac tion. One can argue that it is due to inefficient extraction of the vortexing process. However, the extracted fraction from glass fiber HEPA filters (162 61) following the PAGE 60 60 same vortexing procedure was much higher, demonstrating that vortexing extraction was efficient for regular filter media (Li et al. 2008). High retention capability of the electret test filter can be a reason for the low extracted fractio n due to electrostatic attraction between viral particles and filter media. In the same context, insignificant reaerosolization of the viruses from the test filters is expected. It should be noted that both iodine-treated and untreated filters presumably have similar retention of viruses. In the test filters, the nega tively charged surface of viruses is influenced by an attractive force with the positive resin surface and repulsive force due to negatively charged functional groups on the filter medium. This property of the test filter implies that a filter medium that is depleted of I2 over time can still serve as an effective medium for trapping viruses because it ha s the same attractive and repulsive forces as the untreated filterthe resin surface and by-product iodide ions remain after consumption of the iodine molecules from the triiodide ions. The effect of iodine on the inf ectivity of MS2 collected on the iodine-treated filter is less certain than previously thought, b ecause similar viable recoveries were observed for the iodinetreated and untreated filters; howev er, a strong virucidal effect of I2 was observed in both the VRE of the iodine-treated filter and free I2 residual experiments. This phenomenon can be explained by two possible reasons: (1) shielding e ffect of aggregated particles collected on the filter and (2) high retention cap ability of the test filters. Shielding effect MS2 in suspension is vulnerable to iodine, because the virus is better dispersed in an aqueous medium, whereas in the air it can be aggr egated or encased in other constituents of particles that protect it from iodi ne inactivation. This assertion is supporte d by the SEM images shown in Figure 3-4. Most particle s observed in the tested filter are orders of magnitude larger PAGE 61 61 than a single naked MS2, which can be either the MS2 aggregates or substances with MS2 generated from the nebulizer suspension (virus st ock suspension in the nebulizer contains milk proteins and organic molecules for virus preserva tion). Therefore, infectivity of MS2 can be shielded by the outer layer of the aggregates or by encasement in substances present in the nebulization medium. MS2 aggregation generate d from the nebulizer, which is caused by hydrophobic interactions between neighboring protei n capsids, has been observed by previous studies (Hogan et al. 2004; Balazy et al. 2006). High retention capability of the filter The extracted fractions of both iodine-treated and untreated filters are significantly lower than the other regular filter media due to the e xpected high retention of particles on filter media resulting from electrostatic in teraction between filter media a nd the charged surface of viral particles, as discussed earlier. It should be st ated that this interaction will persist due to the inherent electret property of th e resin-treated surface. Extracte d values close to the detection limit can make the effect of iodine on the virus infectivity indistinguishable. The control experiments carried out in this study with thio sulfate and BSA require that reported data generated in experiments collecting aerosols in aqueous media or on protein gels to measure the biocidal capacity of the iodine-tre ated filter be reexamined to consider the possibility of competition by dissolved I2. Significant support for the previously proposed mechanism of charge-induced cap ture of iodine from bound triiodi de is found in the observation of significant inactivation persisting in a BSA medi um that was able to protect suspended virions from inactivation by impinging I2 vapor. However, toxicity of i odine dissolved in the collection medium is likely to be a competing mechanism in warm environments, and the relative importance of each must be determinedor at least factored into the design and analysis PAGE 62 62 processesat different conditions Data from a different expe rimental approach might not encounter this uncertainty, and the assay is only a surrogate for the goal of the technology enhancing respiratory protection ag ainst bioaerosol transmission of pathogens. Both the medium in the impinger and the protein gel have elements in common with respiratory mucosa, and for a person wearing individual protective gear, the time of transit from filter to mucosal surface is similar. However, competition by water and by pr oteins at the site of impaction might or might not behave the same as in the in vitro system s tested to date. So, the ultimate measure of enhancement of protection by surface-bound iodi neor any other reac tive surface on filter fiberswill require data fr om animal exposure studies. PAGE 63 63 Table 3-1. Removal efficiency of the iodine-treated and untreat ed filters for MS2 aerosols at various environmental conditions in impi ngers containing phosphate buffered saline Virus Concentration (PFU/mL)* Environmental conditions Filter media Challenge Penetration Removal eff. (%)* No.1 1.0 105 4.3 1045.3 102 2.5 102 99.4 0.5 Iodinetreated No.2 1.4 105 5.8 1044.1 102 3.4 102 99.7 0.4 No.1 6.3 104 5.6 1045.0 103 4.4 103 92.4 1.8 Room temp. (23 2 C) Low RH (35 5%) Untreated No.2 3.7 104 1.2 1043.3 103 1.1 103 90.7 2.2 No.1 1.4 105 7.0 104N.D > 99.9995 Iodinetreated No.2 3.0 104 2.5 1043.2 100 2.4 100 99.98 0.05 No.1 3.3 105 1.5 1051.6 104 6.9 103 94.0 3.8 High temp. (30 2 C) Low RH Untreated No.2 9.6 104 3.0 1047.2 103 2.7 103 91.4 4.8 No.1 2.4 104 1.8 1046.7 101 6.9 101 99.8 0.3 Iodinetreated No.2 7.6 103 3.2 1034.2 100 8.8 100 99.8 0.8 No.1 2.3 105 2.4 1051.4 104 1.3 104 93.4 2.1 Room temp. Medium RH (50 5%) Untreated No.2 1.0 105 3.8 1048.9 103 3.5 103 91.3 2.0 The average ( S.D) of five 2-hr trials, Not detected. PAGE 64 64 Table 3-2. The survived MS2 among various MS2 concentrations in the impingers with phosphate buffered saline, thiosulfate solu tion, and bovine serum albumin at various environmental conditions due to released iodine from the filter Virus count (PFU) in the impinger (Average SD) Environmental conditions Collection medium in the impinger Control experimental Survival fraction* 5.6 1030 0 1.1 1041.0 1029.1 10-3PBS 2.3 1058.0 1023.4 10-3Room temp. (23 2 C) Low RH (35 5%) Sodium thiosulfate 1.9 103.9 1021.7 103.9 1029.0 10-1.0 6.3 103.1 10100 5.3 104.5 1038.6 101.4 1011.6 10-3.5 10-4PBS 2.1 105.5 1045.0 102.1 1022.4 10-3.9 10-4High temp. (30 2 C) Low RH Sodium thiosulfate 1.4 103.4 1021.2 103.0 1008.5 10-1.1 10-2 6.2 103.5 10300 6.5 104.1 1034.9 101.9 1017.3 10-4.1 10-4PBS 2.9 105.4 1045.6 102.2 1012.0 10-3.1 10-5Sodium thiosulfate 3.2 103.5 1032.2 103.5 1037.5 10-1.1 10-2 0.3 % bovine serum albumin 1.6 103.7 1029.1 102.3 1025.9 10-1.7 10-1 3 % bovine serum albumin 1.6 103.4 1021.5 103.1 1029.5 10-1.1 10-2 Room temp. Medium RH (50 5%) 6 % bovine serum albumin 1.9 103.6 1021.7 103.4 1029.5 10-1.3 10-2 PFU in the experimental impinger divided by PFU in the control impinger, Phosphate buffered saline PAGE 65 65 Table 3-3. Iodine concentration (mg I2/L)* in the vortexing soluti on at each vortexing time Vortexing Time (min) Filter Media 015 10 Iodine-treated filter 0.62 0.110.98 0.040.91 0.13 0.98 0.08 The average measurement in triplicate Table 3-4. Extracted fraction of MS2 on the iodi ne-treated and untreated filters at various environmental conditions Average SD Environmental conditions Filter media Observed Corrected* Iodine in vortexed solution (mg/L) Iodine-treated 3.4 10-3 1.4 10-32.0 10-2 8.4 10-3 0.93 0.01 Room temp. (23 2 C) Low RH (35 5%) Untreated 3.6 10-2 3.4 10-2 Iodine-treated 3.3 10-3 2.0 10-32.0 10-2 1.2 10-2 0.575 0.007 High temp. (30 2 C) Low RH Untreated 3.3 10-2 2.7 10-2 Iodine-treated 1.2 10-3 5.0 10-46.9 10-3 2.9 10-3 0.76 0.06 Room temp. Medium RH (50 5%) Untreated 5.5 10-3 9.2 10-4 The value was obtained by dividing the observe d values by the surv ival fraction (0.17). PAGE 66 66 A B Figure 3-1. Experimental set-up. A) Viable removal efficiency. B) Physical removal efficiency of the test filters PAGE 67 67 Figure 3-2. The number-based par ticle size distribution of aeroso ls entering and penetrating the filter at RT/LRH Figure 3-3. The survived MS2 aerosols among penetr ated MS2 aerosols from the iodine-treated filter with thiosulfate solution and 3% bovine serum albumin as the collection medium of the impinger at room temperature and medium relative humidity PAGE 68 68 A B Figure 3-4. SEM images of the f ilter at 2700X. A) Unused iodine-tre ated filter. B) Iodine-treated filter after vortexing experiment PAGE 69 69 CHAPTER 4 CHARACTERIZATION OF MS2 BACTERIO PHAGE AEROSOLS INFLUENCED BY RELATIVE HUMIDITY AND SPRAY MEDIUM Objective The objective of the study presented in this ch apter was to characterize viral aerosols by investigating the number of infec tious and total viruses, includi ng infectious and non-infectious, in the ultrafine and submicrometer range. Relati ve humidity and spray medium from which viral aerosols were generated were fact ored into the investigation due to influence of those on the stability or infectivity of viral aerosols. The ul timate goal of this study is to provide information on how viruses are distributed and survive in aer osols under different environmental conditions. Such information is important to a wide range of applications such as development of protection method, respiratory deposition of viral aerosols and consequently risk assessment of airborne pathogens. Materials and Methods Test viruses MS2 is a non-enveloped, icosahedron-shape d, single-stranded RNA with a diameter of 27 34 nm that infects only male Escherichia coli (Stokley et al. 1994; Prescott et al. 2002) MS2 has been used as a simulant for human pathogens of small RNA viruses such as Ebola virus, poliovirus, and rotavirus because of its simila r physical characteris tics including small size and simple structure. Because it is harmless to humans, economical and easy to culture and assay, MS2 has been used as a model microorgani sm in a number of studies: biological defense studies (Belgrader et al. 1998; Kuzmanovic et al. 2003; O'Connell et al. 2006), testing protection device against biological agents (Walker et al. 2004), and detection of microorganisms in environment (Alvarez et al. 2000). It consists of a 3,569 nucleotide genome encoding four PAGE 70 70 proteins a coat protein, a maturation protein, a replicate subunit (or RNA replicase chain), and a lysis protein and 180 copies of the capsid protein (F iers et al. 1976). The MS2 virus stock was prepared by suspending freeze-dried MS2 (ATCC 15597-B1), which contains a small amount of milk proteins and organic molecules for virus preservation, with filtered deionized (DI) water to a concentration of 10910 plaque forming units (PFU ) /mL and stored at 4 C. Experimental design The experimental set-up to investigate the infectious and non-infectious viruses as a function of particle size is s hown in Figure 4-1. The aerosols containing viruses were produced by a Collison nebulizer and dried in the dilution dr yer to remove water content. The resultant aerosol had a polydisperse partic le size distribution (PSD), whic h was characterized by using the scanning mobility particle size r (SMPS), a device that operates as the combination of an electrostatic classifier with a long differential mobility anal yzer (DMA) and a condensation particle counter (CPC), as show n in Figure 4-1 (A). Since the change of PSD over the entire generation is important, the PSD was monitored for 35 min, which was the time needed to conduct the experiment. The voltage applied to the differential mob ility analyzer (DMA) can be tuned to allow only aerosols of a specific size to exit th e electrostatic classifier. The size classified aerosols were subsequently collected in a BioSampler (SKC Inc., Eighty Four, PA, USA) for 5 mins with a flow rate of 4.5 Lpm as shown in Figure 41 (B). The reason for using a flow rate lower than the standard one ( i.e., 12.5 Lpm) is to avoid significant reaerosolization fr om the impinger at the higher flow rate. Because Riemenschne ider et al. (2009) reported insignificant reaerosolization (<1 %) over short sampling peri ods, 5 min of sampling time was selected. The samples in the BioSampler were then analyzed with a plaque assay method (Lee et al. 2009) by PAGE 71 71 inoculating host cells in the samples and pol ymerase chain reaction (PCR) to investigate infectious and total vi ruses, respectively. Since the effect of RH on th e stability or infect ivity of viral aero sols was hypothesized, three RHslow RH (25 5 %, LRH), medium RH (45 5%, MRH), and high RH (85 5%, HRH) were considered by adding dry or humid air in to the dilution dryer. The size distribution function of infectious viruses based on the re sults of the plaque assay method was calculated following Eq. (4-1). t d Q C V C cm PFUp inlet Eff PFU log /3 (4-1) where CPFU is the virus concentration in the co llection medium of the BioSampler, V is the volume of the collection me dium of the BioSampler, CEff is the correction factor for the collection efficiency of the BioSampler for specific particle size, which is adopted from Hogan et al. (2005) and is li sted in Table 4-1, Qinlet is the inlet flow rate of DMA, t is the collection time of the BioSampler, and log dp is the interval of specific pa rticle size range set by the DMA. Three different types of virus suspensions were tested. They were prepared by spiking 0.5 mL of virus stock in 50 mL of filtered sterile DI water, 0.25 % tr yptone in filtered sterile DI water, and artificial saliva. Tr yptone is derived from casein by en zymatic treatment that provides a source of peptides and amino acids for grow ing bacteria. When MS2 was aerosolized with tryptone, the stability of airborne MS2 over a wide range of RH was reported (Dubovi and Akers 1970). To preserve the infectivity of MS2 aerosol s and simulate substances in the air that can contribute to encasement of viruses, the virus suspension was aerosolized with tryptone. The artificial saliva was used to emulate the situa tion where human beings are the source of viral aerosols. Components of the artificial saliva were taken from prior studie s (Veerman et al. 1996; Wong and Sissions 2001; Aps and Martens 2005) a nd are listed in Table 4-2. Regarding the PAGE 72 72 protein components in the artifici al saliva, mucin (one of major components of saliva) was added to the total protein concentration level in sa liva. The mucin-containing saliva is the best substitute of natural saliva in rheological properties, and viscosity and elasticity of this medium are responsible for the protective role of saliv a against desiccation (Vissink et al. 1984). Depending on the virus suspension in the nebulizer, the size of dry aerosol s or droplet nuclei ( dp) can be calculated from the droplet diameter, dd, according to Eq. (4-2) (Hinds 1999c). dp = dd (Fv) 1/3 (4-2) where Fv is the volume fraction of solid content in nebulizer suspension. The volume fraction of solid content for MS2 suspension in DI water, 0. 25% tryptone solution, a nd artificial saliva was 9.9-4, 3.5-3, and 2.1-2, respectively. After complete ev aporation, the particle size of MS2 aerosols generated from DI water, tryp tone solution, and arti ficial saliva was 0.10 dd 0.15 dd, and 0.28 dd, respectively. Seven particle sizes were selected including (1) 30 nm, which is close to the nominal MS2 primary particle size, (2) 230 nm, which is the upper limit of particle size measured by the SMPS when the sample flow of the electrostatic classifier is set at 1.5 Lpm, and (3) 60 nm, 90 nm, 120 nm, 150 nm, and 200 nm, which provides information of intermediate sizes. PCR assay Before submission to PCR analysis, 4 mL samples were concentrated to 280 L by using an Amicon ultracentrifugal device (UFC 810096, Millipore, Bedford, MA, USA) followed by RNA extraction with QIAamp Viral RNA mi ni kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturers in structions and stored at -80 C. A previous study (O'Connell et al. 2006) of real-time fluorogeni c reverse transcriptase (RT-PCR) assays for detection of MS2 was followed for design of primer and probe sequences. The GenBank accession number was PAGE 73 73 NC_001417. The sequences set for RNA replicase chain were selected for testing because it is a critical component in infecti on process and more relevant than other genes. For reverse transcription (RT), 10 L of reac tion mixture prepared from 2 L of 10X RT buffer, 0.8 L of dNTPs, 2 L of reverse primer, 1 L of revers e transcriptase, and 4.2 L of DNase/RNase free water was mixed with 10 L of extracted viral R NA for a total final volume of 20 L. The first RT step was carried out at 65 C for 5 min and immediately quenche d on ice for at least 1 min. The thermal cycling setting for RT was 10 min at 25 C, 2 hrs at 37 C, and 30 secs at 85 C. The RT products (cDNA) were immediately cooled to 4 C. For RT-PCR, 5 L of cDNA was added to 10 L of TaqMan Universal Master Mix (Applied Biosystems), 1.25 L of each primer (f orward and reverse) a nd probe, and 5 L of DNase/RNase free water to a final volume of 25 L. All primers and probes were synthesized by Applied Biosystems. PCR was performed at 50 C for 2 min, then at 95 C for 10 min, followed by 50 cycles of 15 s at 95 C and 60 s at 60 C, on 7900HT fast real-time PCR system (Applied Biosystems). DNase/RNase free water wa s substituted for RNA to prepare the negative control. Results and Discussion MS2 aerosols generated with sterile DI water Figure 4-2 shows the number-based and massbased PSDs for aerosols generated from MS2 suspension in sterile DI water at LRH. Th e size distribution functio n of infectious count obtained by Eq. (4-1) is also presented. Simila r trends of following mass-based PSD are also observed at MRH and HRH, which are presente d in Appendix D (raw data are available in Appendix E). The PSD of aerosols monitored by the SMPS showed negligible variation during experiment indicating constant generation of PSD. Infectivity of MS2 in the nebulizer PAGE 74 74 suspension was analyzed before and after each experiment. Insignificant change in the infectivity proves negligible mechanical stress induced by the aerosolization process on the MS2 infectivity. The plaque assay results were compared w ith the PSD of aerosols to investigate the number of infectious viruses as a function of particle size. Since sterile DI water having 0 PFU/mL (baseline) also generates aerosols, at very low concentrati on, the number of aerosol particles was corrected by subt racting the baseline PSD. Figur e 4-3 shows the number of PFU per particle ( NPFU) as a function of particle size ranging fr om 30 to 230 nm at three RHs. Since the collection efficiency of the BioS ampler depends on the particle size, NPFU was corrected for that given particle size using the collection factor listed in Table 4-1. The theoretical NPFU ( NTheo PFU) was calculated from the volume fraction of inf ectious MS2 in the solid content of the spray medium for the given particle si ze, according to Eq. (4-3). 3 2 26MS MS dp PFU Theod F V N (4-3) where Vdp is the volume of the droplet nuclei, FMS2 is the volume fraction of infectious MS2 in the solid content of the spray medium obtained from the plaque assay for MS2 stock suspension (volume of infectious MS2/volum e of freeze-dried MS2 stock), and dMS2 is the nominal size of MS2 (27.5 nm). As shown in Figure 4-3, more PF U was enumerated at all particle sizes at LRH compared to other RHs. Possible reasons for this dependence on RH in clude stability of MS2 aerosols at LRH and/or enumeration of more vi ruses at LRH than other RHs when the same particle size is compared. After wet dissemination, particle size changes due to evaporation, the rate of which depends on the surrounding RH. Th erefore, for any same registered particle size set by the classifier at three RHs, there are mo re MS2 virions at LRH than at MRH and HRH due PAGE 75 75 to different evaporation rate. At the same RH, NPFU increased as particle size increased with similar trend to NTheo PFU, implying that the increase of NPFU follows the increase of particle volume. This observation can be verifi ed by conducting regression analysis of NPFU as the dependent variable and particle size (dn) as the explanatory variable. The n value at three RHs, which is presented as the slope of least squares regression line (ln( NPFU) vs. ln( dp)) in Table 4-3, can be compared to support this observation. As shown, the n value of NPFU was in the vicinity of 3 at all three RHs, im plying that the increase of NPFU followed the increase of particle volume. Hogan et al. (2005) examined 25 120 and 300 nm MS2 aerosols and their results indicated increasing NPFU as particle size increased, although th ey did not report th e particle volume relationship. Total viruses, including infectious and non-infectious viruse s, of a given particle size were investigated by quantifying RNA in the aqueous collection medium after considering the correction factor for collection efficiency of the BioSampler. The threshold cycle ( CT) value of the sample from RT-PCR was compared w ith a standard curve obtained by plotting CT value for serial dilutions of commercia lly available MS2 RNA (Roche Diagnostics, Indianapolis, IN, USA) against the experimental RNA amount. Using 1.0 106 g/mol as the molecular weight of MS2 RNA (Kuzmanovic et al. 2003), the number of MS2 RNA in the samples was calculated with the conservative assumption that one MS2 R NA represents one MS2 vi rion. The number of MS2 RNA per particle ( NRNA) was then determined by dividing it by the total aerosol particles measured by the CPC following Eq. (4-4): Eff RNAC particles of aerosol number molecules mol mol g ng g Sample the in ng RNA N 23 6 910 02 6 1 1 10 0 1 ) / 10 ( ) ( (4-4) PAGE 76 76 It should be noted that NRNA includes any fragment of MS2 R NA containing the target sequence for PCR as well as infectious RNA. Biodegradat ion of RNA having target sequence can lead to underestimated PCR results; specia l care of storage and sample ha ndling is needed to prevent underestimates. PCR analysis was conducted for two experimental sets and for selected particle sizes. Table 4-4 shows NRNA for several particle sizes at three RH s as well as the theoretical value of NRNA. Similar to NTheo PFU, theoretical NRNA ( NTheo RNA) was also calculated using Eq. (4-3) except that total MS2 instead of infectious MS2 was considered. In the equation, FMS2 was calculated for total MS2 with the assumption that the freeze -dried MS2 stock is mainly composed of MS2 particles with negligible impurity. As shown in Table 4-4, the enumeration of MS2 RNA in a given particle size increased as RH decreased due to more contri bution of solid content to the particle size. At the same RH, the value gene rally increased as particle size increased. The increased rate of NRNA at three RHs was generally much less than that of NTheo RNA, which can be confirmed by the same regression an alysis applied for comparison of NPFU at three RHs. Table 4-5 lists the n value (the slope of the regr ession analysis) at three RHs. As shown, the presence of total viruses ( NRNA) in aerosol particles increased in proportion to particle surface area ( n = 2) or an even lower dimension. Th e difference between experimental NRNA and NTheo RNA can be attributed to the discontinuous di stribution or generation of MS2 particles in the suspension. Whereas NTheo RNA was calculated with the assumption that MS2 particles are uni formly dispersed, in reality the distribution of MS2 particles in the nebulizer suspension is not uniform. The presence of MS2 aggregates in the nebuliz er suspension caused by hydrophobic interactions between neighboring protein capsids has been obse rved in previous studies (Hogan et al. 2004; Balazy et al. 2006). Therefore, MS2 virions can be present in aerosols as individuals, as PAGE 77 77 aggregates or attached to the su rface of the solid content. At the same time there are particles that contain no MS2. The stability of MS2 aerosols was investigated by comparing NPFU/NRNA ( i.e., infectious MS2/total MS2) of select particle sizes at three RHs as shown in Table 4-6. NPFU in a unit RNA was significantly higher at LRH th an at MRH and HRH (one-way ANOVA, p -value < 0.05), indicating stability and preserva tion of MS2 infectivity in aeros ols at LRH. No significant difference was observed between the valu e at MRH and HRH (unpaired Students t -test, p -value > 0.05), indicating similar survival capacity. This observation can be attributed to the increase in air to water interface at increased RHs, which results in the exposure of aerosols to unbalanced force leading to a decrease of NPFU (Adams 1948). NPFU/NRNA generally increased as particle size increased at MRH and HRH in spite of the adve rse effect at increased RHs. This result demonstrates the shielding effect of bigger particles. Indeed, MS2 aerosols, which are less stable at 50% and 85% than at 25% RH can be protected by forming a ggregates to reduce exposure to the adverse influence of increased RH. Meanwhile, at LRH, NPFU in a unit RNA differed insignificantly among the various particle sizes investigated. This shows that, without the adverse effect of RH, shielding due to aggr egation decreases in importance to survival. MS2 aerosols generated from tryptone solution Experiments were also conducted with tyrptone solution as the aeros olization medium. As shown in Figure 4-4 for LRH, the presence of tryptone in the nebulizer suspension shifted the PSD towards the bigger particle size range compar ed to the MS2 aerosols generated from sterile DI water as Eq. (4-2) predicts. The PSD of infectious viruses was between numberand massbased PSD, i.e. its n value was between 2 and 3 as shown in Table 4-3. At other RHs, a similar phenomenon was also observed (Appendix D). The NPFU of a given particle size was also PAGE 78 78 calculated by following the same equation used fo r sterile DI water (shown in Figure 4-5). Similar to the MS2 aerosols generated with sterile DI water (shown in Figure 4-3), NPFU increased as particle size increased. However, the values at three RHs increased less with increasing particle size than NTheo PFU ( n = 3), and were also lower than those for sterile DI water (Table 4-3). It is plausibl e that the abundance of tryptophan in tryptone induces hydrophobic interaction with MS2 protein and also provides surface for MS2 to re side on or attach to. This phenomenon can cause NPFU increase in proportion to surfac e rather than to volume. It should also be noted that NPFU was significantly lower than that generated from sterile DI water at LRH. The reason for this phenomen on is the contribution of tryptone to the solid content of droplet nuclei, which leaves less room for MS2. This contribution can be verified by analyzing NRNA in the samples. By using the calcula tion used for MS2 aerosols generated from sterile DI water, NRNA of select particle sizes at three RH s was calculated (shown in Table 4-7). Clearly, NRNA was significantly smaller than that of MS2 aerosols generated from DI water (Table 4-4), due to the significan t solid fraction resulting from the presence of tryptone. The NRNA of a given particle size was higher at LRH than at MRH and HRH due to increased solid contents; meanwhile, insignificant differen ce was observed between MRH and HRH. Table 4-8 presents NPFU/NRNA at three RHs. At LRH, NPFU/NRNA shows similar values among different particle sizes. Th e result demonstrates that when viruses are not exposed to the adverse effect of increased RH, the presence of tryptone exerts no protective effect. NPFU/NRNA at HRH was significantly higher th an that at LRH and MRH, as well as at HRH for MS2 aerosols generated from sterile DI water. This observatio n can be explained by the encasement effect of tryptone for MS2 aerosols in the hostile condition of HRH. A si milar study demonstrated high recovery of MS2 aerosols at all RHs ranging from 20 to 80% due to the protective effect of PAGE 79 79 tryptone (Dubovi and Akers 1970). With in the same context, increased NPFU/NRNA for MS2 aerosols generated from tryptone so lution than that for MS2 aerosols generated from suspensions in DI water was expected at MRH. However, as seen in Tables 4-6 and 4-8, insignificant increase was observed. Th is observation, along with th e significant decrease of NPFU compared to that for sterile DI water at LRH, suggests an adverse effect of tryptone at LRH and MRH, rather than a protective effect. This result can be attributed to the supersaturated condition of tryptone in droplet at LRH and MRH. Although this observation does not agree with the Dubovi and Akers (1970) study in the aspect that they ob served high recovery of MS2 at LRH and MRH, Trouwborst and de Jong (1973) de monstrated that phenylalanin e does not exert a protective effect for MS2 aerosols under supersaturated cond itions. They mentioned that crystals or the process of crystallization can be deleterious to MS2 aerosols. As the RH keeps decreasing, droplets may reach the crystalli zation RH (CRH), which is the maximum RH at which solutes maintain the aqueous phase withou t experiencing crystallization at a supersaturated condition. The CRH is always below the deliquescence RH (D RH). It was reported that the DRH and CRH of ammonium sulfate are 80% and 40%, respec tively (Seinfeld and Pandis 1998). Also, some common components of ambient aerosols have a DRH between 70% and 85%. Although the CRH of these components was not reported, it is reas onable to expect that th e value is similar to that of ammonium sulfate unless the species are not hygroscopic. Within the same context, the CRH of various components of the spray medi um can be around 40%, which is about the MRH investigated in this study. MS2 aerosols generated with artificial saliva Figure 4-6 shows the PSD of number-based, mass-based, and infectious counts as a function of particle size at LRH. Apparently, the PSD was shifted to an even bigger particle size PAGE 80 80 range compared to the PSD generated from tr yptone solution due to the increased volume fraction of solid materials in the nebulizer suspension. The PSD of infectious viruses followed a lower order dependence on dimension, between number and area distributions, as shown in Table 4-3. The results at other RHs show a sim ilar pattern and are presented in Appendix D. Figure 4-7 shows NPFU as a function of particle size at three RHs. Compared to the MS2 aerosols generated from tryptone solution and sterile DI water, there was less increment as particle size increased at three RHs. There are two possible reasons for this phenomenon: (1) negligible shielding effect of bigger particles due to insuffici ent amount of MS2 virus to be aggregated, and (2) adverse effect of saliva co mponents on viral aerosols. In terms of the amount of MS2 viruses, the NRNA values for MS2 generated from artificial saliva are similar to those for tryptone soluton, as shown in Tables 47 and 4-9. Since the la tter presented a shielding effect, MS2 aerosols generated from arti ficial saliva should have sufficient NRNA to present a shielding effect of aggregates. The fact that NPFU is low implies that MS 2 virions in aerosols do not aggregate well to achie ve a shielding effect. To address th is issue, one should recall that the artificial saliva used in this study is a mucin-cont aining medium. Mucin has an oligosaccharide chain containing numerous hydropho bic regions, which are respons ible for its sticky property (Mehrotra et al. 1998; Zalewska et al. 2000). In a later study (H abte et al. 2006), it was observed that mucin aggregates HIV-1 (human immunodefici ency virus type 1) leading to an enhanced filtration through 0.45 m pore size cellulose acetate filters. Therefore, it can be inferred that mucin induces hydrophobic interac tion with MS2 protein, thus reducing MS2 aggregation by itself. The lack of shielding effect of aggregat es is verified by the lower slope value shown in Table 4-3. PAGE 81 81 The negligible increase of NPFU as particle size increases can also result from the adverse effect of saliva components. The adverse effect of saliva on the stability of viral aerosols has been reported in a previous study (Barlow and Donaldson 1973). They observed that foot-andmouth disease viral aerosols were more unstable when generated from bovine salivary fluid than from cell culture fluid at HRH, a nd they postulated the presence of an inactivating factor in the saliva as the reason for instability of viral aerosols. In later studi es (Fox et al. 1988; Bergey et al. 1994; van der Strate et al. 2001; Ha rtshorn et al. 2006), an antiviral effect of saliva on HIV-1 and influenza A virus was observed a nd some proteins of saliva such as lactoferrine, agglutinin, and mucins were proven to be the inactivating factors. Table 4-10 shows NPFU/NRNA at three RHs. The values at LRH and MRH were similar to those for MS2 aerosols generated from tryptone solution, indicating a similar adverse effect. At HRH, the values were lower than those from the tryptone medium. Both inactivation effects from salivary components and from the air/water in terface can be factors. The protective effect of tryptone at HRH was not obser ved for artificial saliva, showi ng again the adverse effect of saliva components. However, no synergistic effect of these two factors was observed since the NPFU/NRNA values were similar to those for MS2 aerosols generated with DI water, which were adversely influenced only by th e air/water interface. Distribution of MS2 in aerosol particle s generated from different spray media As presented in Tables 4-3 and 4-5, the di stribution of MS2 including both infectious and total (infectious and non-inf ectious) viruses along the aerosol size ranging from 30 to 230 nm was investigated. The n values for NRNA and NPFU for aerosols generated from DI water and from tryptone solution were different, althou gh the difference was less for tryptone solution than for sterile DI water. On the other hand, MS2 aerosols generated from saliva showed a much PAGE 82 82 smaller n value for both NPFU and NRNA. Since NPFU represents only infectious viruses while NRNA includes fragments of nucleic acid and non-infec tious viruses, and inf ectious viruses, these two values can be quite different in the presence of other substances. To assess the influence of spray media on NPFU and NRNA, two-way ANOVA analysis was conducted. For NRNA, the n value showed negligible difference among the media ( p -value > 0.05). Meanwhile, the n value of NPFU exhibited a significantly different increase rate ( p -value < 0.05); it decreased as solid material in the spra y medium increased in the order DI water (2.9), tryptone solution (2.4), and artifici al saliva (1.1). Note that the values presented in parenthesis are averaged at three RHs. This difference between NRNA and NPFU, and also among spray medium for NPFU can be attributed to a combination of several factors, including shielding and encasement effects. The infectious viruses ( NPFU) protected by shielding or encasement effects increase generally in proportion to volume distribution as particle size increases. Regarding MS2 aerosols generated from artificial saliva, the adverse effect of saliva and negligible shielding effect contribute to the similar results between NRNA and NPFU and to a much smaller n value than the other spray media. The PSD of infectious viruses (PFU/cm3, shown in Figures 4-2, 4-4 and 4-6) for different spray media showed that infectious viruses are more abundant from a relatively pure virus suspension (sterile DI water) than from so lid-containing spray medi a (tryptone solution and artificial saliva) at LRH. It s hould be emphasized that the size ra nge for this observation is from 30 nm to 230 nm. If the window were expanded to include bigger particle sizes, it is possible that more MS2 from a solid-containing spray me dium would be enumerated than that from a relatively pure virus suspension. This phenomenon is s upported by the theory of aerosol nebulization (Eq. 4-2). As shown, the aerosol diameter is determined by the droplet diameter PAGE 83 83 and volume fraction of solid materials in the spra y medium. Figure 4-8 illu strates the theoretical shrinkage of droplets to droplet nuclei for MS2 aerosols generated from these three different nebulizer suspensions. The dropl et nuclei resulting from the same droplet get smaller as the solid fraction in the nebulizer suspension decrease s. For instance, a droplet of 2000 nm shrinks to a nucleus about 200 nm from sterile DI wate r, while the correspondi ng droplet nuclei from tryptone solution and from artificia l saliva are about 300 nm and 560 nm, respectively. Since the amount of MS2 stock suspension in the nebulizer is the same for all three spray media, the total aerosols of MS2 aerosols generated from the nebuliz er should also be the same. Therefore, it is reasonable to expect that aerosols of 300 nm and 560 nm generated from solid-containing spray media contain similar amounts of NRNA and NPFU to that observed for 200 nm aerosols generated from DI water. DISCUSSION Regarding the effect of spray medium, two fundamental questions arise: (1) does adding tryptone really help preservation of MS2 aerosol s or disseminate MS2 aerosols more effectively than from DI water? and (2) does saliva help re duce the hazard of viral aerosols? The latter question is of particular interest in relation to recent human cases of influenza A (H1N1) virus infection and its rapidly evolving situation. The presence of tryptone in the spray medium results in two contrary phenomena. In a dry environment, tryptone can be deleterious to MS2 due to crystallizat ion under supersaturated conditions in aerosols. Meanwhile, sensitive MS2 at increased RHs can be protected by the encasement effect of tryptone, and thus enumerat ed in a relatively larger numbers. This statement can be verified by comparing the result s with those from sterile DI water. Although the number of total viruses ( NRNA) in aerosols decreased due to the contribution of tryptone to the PAGE 84 84 aerosol size, the number of infectious viruses ( NPFU) at HRH was similar to that from sterile DI water. In other words, at the optimal condition fo r the stability of viral aerosols (LRH), the most effective way to disseminate viral aerosols is to use a pure virus suspension. On the other hand, the presence of a protective material in the spray medium is a key factor for the spread of viral aerosols at sensitive conditions. In addition to the stability of viral aerosols and the presen ce of protective materials, the effectiveness of spreading viruses depends on the aerosol size. It was already addressed earlier that the size of droplet nuclei is affected by the solid fraction in the spray medium and thus the PSD of aerosols generated from different spray me dia will be present in different size ranges. Depending on the particle size c onsidered, the effective viru s suspension for disseminating viruses varies. If a bigger particle size is de sired, a solid-containing vi rus suspension will be a more effective way to spread viruses than a rela tively pure virus suspension. This study proves that both environmental factors ( e.g., RH) and substances in th e virus suspension play a significant role in the fate of viral aerosols. Furthermore, these factors can be protective or deleterious, depending on the combination. For MS2 aerosols generated from artificial saliv a, the adverse effect of salivary protein was observed. Although certain viruses including ad enovirus and vaccinia viru s, are not or little affected by salivary proteins (Ber gey et al. 1993; Malamud et al. 1993), the antimicrobial role of saliva has been extensively observed. Hence, in the scenario that human beings are the source of viral aerosols, the consequence of spread of viral aerosols can be less profound than expected because of the resulting lower numbe r of infectious viruses. As di scussed earlier, the presence of solid materials (saliva component s) can reduce the amount of vi rions or lower the degree of aggregation in aerosols compared to the pure vi rus suspension. From these observations for PAGE 85 85 tryptone and saliva, it can be informed that both concentration and nature of solid materials dissolved in the spray medium determine the si ze and fate of viral aer osols at any given RH. As discussed previously, three concerns relating to specific ch aracteristics of viruses are small particle size, shielding effect, and encasem ent effect of substances. We observed both a shielding effect of aggregates and an encasemen t effect by the presence of inert materials for 200 nm viral aerosols. It should be emphasized th at aerosols of this size are small enough to reach the alveolar region of the l ungs, and inhalation of one such si ngle particle can easily attain the minimum infectious dose of virus with enha nced shielding and the encasement effect. For example, the NPFU resulting from the penetration of a single 200 nm particle through a filter or respirator is equivalent to the NPFU resulting from the penetration of 100 30 nm particles of MS2 generated from DI water at HRH. Although the current study char acterized one specific species (MS2 bacteriophage), general characteristics applicable to other viral ae rosols can be deduced fr om our findings. The shielding effect of small aggregates is a comm on characteristic of gene ral viruses because of their tiny primary particle size and aggregated air borne state. In addition, as observed in the encasement effect of tryptone, inert materials ( e.g., dust in air or substances generated with viruses) can exert a protective influence on vira l aerosols in adverse conditions. These two general properties can co ntribute to the survival of viruse s in otherwise hostile circumstances ( e.g., sensitive RH and temperature) or even inactiv ation treatments, and to subsequent initiation of infectivity and transmission of disease. PAGE 86 86 Table 4-1. Collection efficiency of the BioSampl er for select particle sizes adopted from Hogan et al. (2005) Particle diameter (nm) Collection efficiency (%) 30 14 60 8 90 5 120 4 150 4 200 4 230 5.4 Table 4-2. Components of artificial saliva Components Content Components Content MgCl2 7 H2O 0.04422 g (NH2)2CO 0.1212 g CaCl2 H2O 0.1288 g NaCl 0.876 g NaHCO3 0.42 g KCl 1.0416 g 0.2 M KH2PO4 7.7 mL Mucin 3 g 0.2 M K2HPO4 12.3 mL Tissue culture medium (DMEM) 1 mL NH4Cl 0.108 g Water 979 mL KSCN 0.194 g pH 7 PAGE 87 87 Table 4-3. Slope of least squares regression line for NPFU vs. particle size for different MS2 suspensions at three relative humidities Slope of least squares regression line ( R2) MS2 suspension in Nebulizer Low RH Medium RH High RH DI water 2.8 (0.8)3.0 (0.9)2.9 (0.8) Tryptone solution 2.4 (0.9)2.9 (0.9)2.1 (0.8) Artificial saliva 1.4 (0.8)1.4 (0.9)0.6 (0.8) Table 4-4. NRNA for MS2 aerosols generated from sterile DI water at three relative humidities NRNA Low RH Medium RH High RH Particle diameter (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 NTheo RNA 30 0.97 0.94 0.66 0.93 N/A 0.05 1.3 60 3.22 N/A* 2.20 N/A 4.04 N/A 10 90 9.72 1.62 N/A 0.38 4.08 0.99 35 120 16.32 8.09 12.17 0.49 9.63 0.64 82 150 39.50 N/A 18.25 N/A 12.52 N/A 160 200 51.17 20.69 31.86 14.11 12.11 2.67 380 230 64.96 N/A 52.26 N/A 3.56 N/A 580 Not available PAGE 88 88 Table 4-5. Slope of least squares regression line for NRNA vs. particle size for different MS2 suspensions at three relative humidities. Slope of least squares regression line ( R2) Low RH Medium RH High RH MS2 suspension in Nebulizer Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 DI water 2.1 (0.9) 1.6 (0.8)2.1 (0.9)1.0 (0.2)1.1 (0.8) 2.0 (0.9) Tryptone solution 1.8 (0.9) 1.9 (0.9)1.4 (0.6)1.5 (0.8)2.9 (0.9) 1.3 (0.7) Artificial saliva 1.4 (0.9) 1.5 (0.9)0.9 (0.9)1.3 (0.8)1.1 (0.8) 1.0 (0.8) Table 4-6. NPFU / NRNA for MS2 aerosols generated from st erile DI water at three relative humidities Log ( NPFU/NRNA) Low RH Medium RH High RH Particle diameter (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 30 -5.1 -5.4-7.5-6.6N/A -6.5 90 N/A N/AN/AN/A-6.6 -6.2 120 -5.5 -5.7-6.9-5.7-6.4 -6.1 200 -5.2 -4.4-5.7-6.1-6.0 -5.9 PAGE 89 89 Table 4-7. NRNA for MS2 aerosols generated from tryptone solution at three relative humidities NRNA Low RH Medium RH High RH Particle dia. (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 NTheo RNA 30 0.18 0.58 0.07 0.26 N/A 0.45 0.38 60 0.39 N/A 0.19 N/A N/A N/A 3.1 90 0.60 N/A 0.39 N/A 0.27 0.50 10 120 2.80 4.91 1.65 1.07 0.46 1.98 25 150 3.79 N/A 1.45 N/A 1.19 N/A 48 200 5.65 27.73 2.38 6.45 2.61 6.71 110 230 5.46 N/A 0.45 N/A 3.80 N/A 160 Table 4-8. NPFU / NRNA for MS2 aerosols generated from tr yptone solution at three relative humidities Log ( NPFU/NRNA) Low RH Medium RH High RH Particle dia. (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 30 -6.1 -6.2-6.5-6.5N/A -5.7 90 N/A N/AN/AN/A-4.7 -5.4 120 -6.8 -6.1-6.3-6.2-4.3 -5.5 200 -5.7 -6.2-5.4-5.7-4.9 -5.1 PAGE 90 90 Table 4-9. NRNA for MS2 aerosols generated from artificial saliva at three relative humidities NRNA Low RH Medium RH High RH Particle diameter (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 NTheo RNA 30 0.3 0.3 0.4 0.4 0.4 0.6 0.05 120 2.3 2.4 1.1 1.3 1.0 1.3 3.8 200 5.0 6.2 2.5 6.1 4.3 5.2 17 Table 4-10. NPFU / NRNA for MS2 aerosols generated from artificial saliva at three relative humidities Log ( NPFU/NRNA) Low RH Medium RH High RH Particle diameter (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 30 -6.6 -6.2-6.3-6.4-5.7 -6.1 120 -6.9 -6.5-5.8-6.2-5.9 -6.1 200 -6.4 -6.9-6.0-6.3-6.4 -6.5 PAGE 91 91 Figure 4-1. Conceptual schematic of the experi mental set-up: A) Measur ement of particle size distribution; B) Collection of vira l aerosols of selected size. PAGE 92 92 Figure 4-2. Particle size distribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aerosol s generated from ster ile DI water at low relative humidity. Figure 4-3. NPFU for MS2 aerosols generated from sterile DI water at three relative humidities. Data shown are the mean of three repetiti ons with error bars re presenting standard error. PAGE 93 93 Figure 4-4. Particle size distribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aeros ols generated from tryptone solution at low relative humidity. Figure 4-5. NPFU for MS2 aerosols generated from tryptone solution at three relative humidities. Data shown are the mean of three repetiti ons with error bars re presenting standard error. PAGE 94 94 Figure 4-6. Particle size distribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aerosol s generated from artificial saliva at low relative humidity. Figure 4-7. NPFU for MS2 aerosols generated from artificial saliva at three relative humidities. Data shown are the mean of three repetiti ons with error bars re presenting standard error. PAGE 95 95 Figure 4-8. Theoretical droplet nuclei diameter as a function of droplet diameter for different nebulizer suspnesions at low relative humidity. PAGE 96 96 CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS Both treated and untreated filters exhibited high viable removal efficiency (> 99.996%) for bacterial spores in various environmental c onditions with negligible variation in pressure drop. This great performance of test filters did not deteriorate over the experimental duration ( i.e. 10 hr or 4 hr). Viability of spores collecte d on the filter was investigated by extracting them from the filter and presented as the survival fr action. A higher survival fraction on the untreated filter than that on the treated filter was reported at RT/LRH. However, the survival fraction of treated filters at RT/HRH and HT/HRH was similar to that of untreated filt ers tested at RT/LRH. Loss of iodine by sublimation and dissoluti on at HT and HRH was responsible for the indifference. As electret filter media, both treat ed and untreated filters presented high retention capability for negatively charged bacterial spores and thus reduced potential hazard from the release of spores from the media. For the viral aerosol filtration experiment, ne w filter media different from those used in the bacterial spore experiment were used. The iodine-treated filter presented high removal efficiency for viral aerosols with low breathing resistance (significantly lo wer pressure drop than the NIOSH regulation) under various environmen tal conditions. Both treated and untreated filters presented similar extracted fractions, indi cating insignificant difference in the infectivity of viruses on both filters. This observation can be attributed to the shielding effect of MS2 aggregates on the filter and/or high retenti on capability of the filter due to electrostatic interaction between charged filter media and viral particles making any difference indistinguishable. Meanwhile, it demonstrates again high retenti on capability that can minimize reaerosolization and prevent air filt ers to be a potential source of mi crobial contamination. As an inactivation mechanism of the iodine -treated filter, transfer of a lethal dose of iodine from the PAGE 97 97 filter to the MS2 aerosols during it s flight through the filter, wh ich was proposed previously, was verified. By comparing the experimental result s of BSA and thiosulfate solution, we concluded that reaction of MS2 in the collection medium of the sampling device with iodine released from the filter was also occurring as a competing inactivation mechanism. This realization requires that these two inactivat ion pathways be factored in the design of the assessment methodology and interpretation of its results. After completing these studies we learned that the threshold iodine concentration for inactivation is low ~ 0.5 ppm in water as disclosed by Triosyn Corp. (Messier 2009)so the concentrations of I2 at which we operated are large enough that the experimental results of the impinger with PBS we re obscured by the free i odine in the collection medium of the impinger. Specifically, measured c oncentrations of released iodine collected with the bioaerosols in the impinger were sufficient to inactivate mi crobes collected in the impinger and to obscure the inactivation pr ocess of iodine on the microbes. In the present experimental configuration, it takes only a fe w milliseconds for microbes to pene trate the iodine-t reated filter before being collected in the impinger, indicat ing a transient reaction time of microbes with iodine in the air phase. Theref ore, even if microbes penetrating the iodine-treated filter accumulate iodine molecules, they can still be viable unless iodine in the surface of microbes work its way in. The experimental result of th iosulfate solution presenti ng survival of microbes penetrating the iodine-treated filt er implies that inactivation process of iodine is not completed in such a short time and must be re latively slow on that time scale. In this context, one cannot exactly interpret the experimental result that BSA allows inactivation of ha lf of penetrating MS2, but one can conclude that some transfer of loosely bound iodine to BSA from aerosols occurs, allowing insufficient reaction time for the labile iodine to inactivate the surviving fraction of microbes. PAGE 98 98 The infectious and total MS2 viruses as a f unction of aerosol size in the ultrafine and submicrometer size range, influenced by relative humidity and spray medi um, were investigated with bioassay and PCR analysis, respectively. Both infectious and total viruses increased as particle size increased although th e increase rate varied dependi ng on several factors such as protective effect and nature of solid content in spray medium. With the stability at LRH and greater solid fraction, the number of infectious viruses was significantly higher at LRH compared to MRH and HRH for MS2 aerosols from DI water. The sensitivity of MS2 to increased RH can be attributed to the unbalanced force of air-water interface. In the presen ce of solid content in viral aerosols (tryptone and artifi cial saliva), the enumeration of MS2 in aerosols decreased due to greater contribution of solid contents to the ae rosol size. The shielding effect of aggregates and encasement effect of tryptone resulted in enhanced MS2 infectivity than expected one by reducing contact of viral aerosols to the adverse factor such as e nvironmental conditions. On the other hand, artificial saliva exer ted adverse effect on the infectivity of viral aerosols and yielded negligible shielding effect. Th e present study demonstrates that even one single aer osol particle can have sufficient infectious virions to exceed the minimum infectious dose due to shielding and encasement effect. It is th erefore critically important to develop new technologies that can more effectively protect the public from airborne vi ral pathogens. Topics for future research in this area include investigation of the effect of the presence of foreign aerosols on the performance of the iodi ne-treated filter and on the inactivation process of iodine on microbes. The presence of forei gn aerosols may hinder the exertion of biocidal effect by resulting in either maski ng of the iodine-treated site w ith these particles or parasitic consumption of oxidizing equivalents. Furthermor e, these substances can serve as nutrients for the growth of collected microorganisms, eventua lly resulting in the i nhalation of bioaerosols PAGE 99 99 from re-entrainment. Full evaluation of such a condition will determine its application to diverse scenarios. Clarification of inactivation mechanis ms for airborne biologica l agents after transit through the iodine-treated filter should be inves tigated, to identify transport mechanisms and reaction pathways of iodine that operat e on the time scale of my experiments. PAGE 100 100 APPENDIX A RAW DATA OF BACTERIAL SPORE EXPERIMENTS Table A-1. Impactor results at room temperature and low relative humidity Iodine-treated filter 1 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.4 34 0 1 1248 40 3.4 32 0 2 1812 60 3.4 32 0 3 3096 80 3.5 32 0 4 5712 100 3.5 33 0 5 28068 120 3.5 32 0 6 58248 Experiment 2. Room Temperature: 23 2 C 20 3.5 36 0 1 888 40 3.6 32 0 2 1860 60 3.5 32 1 3 2724 80 3.5 33 0 4 4920 100 3.5 33 0 5 35616 120 3.5 33 0 6 48552 Experiment 3. Room Temperature: 23 2 C 20 3.4 38 0 1 960 40 3.4 33 1 2 1572 60 3.5 32 1 3 2904 80 3.4 33 0 4 11628 100 3.4 33 0 5 44580 120 3.6 32 0 6 44304 Experiment 4. Room Temperature: 23 2 C 20 3.4 38 1 1 792 40 3.4 33 0 2 936 60 3.5 32 0 3 1884 80 3.4 33 0 4 3024 100 3.4 33 0 5 44580 120 3.6 32 0 6 35616 Experiment 5 Room Temperature: 23 2 C 20 3.6 35 0 1 408 40 3.6 34 0 2 936 60 3.6 32 0 3 1428 80 3.7 32 0 4 3228 100 3.7 33 0 5 42960 120 3.6 34 0 6 29520 CFU is the number of microor ganism normalized to 120 minutes. PAGE 101 101 Iodine-treated filter 2 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.0 34 0 1 552 40 3.0 33 1 2 612 60 3.0 33 0 3 1212 80 3.0 33 0 4 2388 100 3.0 33 0 5 31344 120 3.2 33 0 6 44016 Experiment 2. Room Temperature: 23 2 C 20 3.0 40 0 1 792 40 3.0 38 0 2 948 60 3.0 34 0 3 1644 80 3.0 34 0 4 2616 100 3.0 33 0 5 33372 120 3.2 33 0 6 25884 Experiment 3. Room Temperature: 23 2 C 20 3.0 38 0 1 360 40 3.1 36 0 2 432 60 3.0 33 0 3 1008 80 3.0 32 1 4 2064 100 3.0 32 0 5 30192 120 3.0 32 0 6 31200 Experiment 4. Room Temperature: 23 2 C 20 3.0 39 0 1 264 40 3.0 38 0 2 564 60 3.0 35 0 3 780 80 3.0 33 0 4 2088 100 3.0 33 0 5 30144 120 3.1 33 0 6 25944 Experiment 5 Room Temperature: 23 2 C 20 3.0 39 0 1 516 40 3.0 37 0 2 900 60 3.0 34 0 3 1200 80 3.0 33 0 4 2484 100 3.0 32 0 5 34548 120 3.3 32 0 6 31752 PAGE 102 102 Iodine-treated filter 3 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.3 39 0 1 204 40 3.3 34 0 2 420 60 3.4 33 0 3 600 80 3.4 32 0 4 1380 100 3.4 32 0 5 25536 120 3.4 32 0 6 21192 Experiment 2. Room Temperature: 23 2 C 20 3.4 40 0 1 492 40 3.4 35 0 2 552 60 3.4 35 0 3 1080 80 3.4 34 0 4 2028 100 3.4 33 0 5 27624 120 3.4 32 1 6 26100 Experiment 3. Room Temperature: 23 2 C 20 3.4 40 0 1 168 40 3.4 40 0 2 348 60 3.4 37 0 3 600 80 3.4 36 0 4 1488 100 3.4 35 0 5 25620 120 3.4 32 0 6 22680 Experiment 4. Room Temperature: 23 2 C 20 3.4 39 0 1 564 40 3.4 36 0 2 1104 60 3.4 34 0 3 1800 80 3.4 34 0 4 3408 100 3.4 33 0 5 30468 120 3.4 35 0 6 40368 Experiment 5 Room Temperature: 23 2 C 20 3.4 40 0 1 360 40 3.4 40 0 2 660 60 3.4 37 0 3 1116 80 3.4 36 0 4 2400 100 3.4 35 0 5 31860 120 3.4 35 0 6 35436 PAGE 103 103 Untreated filter 1 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.0 38 0 1 720 40 3.0 36 0 2 828 60 3.0 34 0 3 1260 80 3.0 33 0 4 2472 100 3.0 33 0 5 36264 120 3.0 33 0 6 33324 Experiment 2. Room Temperature: 23 2 C 20 3.0 39 0 1 684 40 3.0 37 0 2 1008 60 3.0 34 0 3 1632 80 3.0 33 0 4 3060 100 3.0 33 0 5 42960 120 3.0 32 0 6 37884 Experiment 3. Room Temperature: 23 2 C 20 3.0 39 0 1 660 40 3.0 34 1 2 912 60 3.0 33 0 3 1380 80 3.0 33 0 4 2700 100 3.0 33 0 5 32292 120 3.2 33 0 6 26484 Experiment 4. Room Temperature: 23 2 C 20 3.0 39 0 1 252 40 3.0 35 0 2 396 60 3.0 35 0 3 492 80 3.0 34 0 4 1560 100 3.0 34 0 5 24672 120 3.2 33 0 6 20604 Experiment 5 Room Temperature: 23 2 C 20 3.0 40 0 1 408 40 3.0 38 0 2 936 60 3.0 36 1 3 1428 80 3.0 34 0 4 3228 100 3.0 33 0 5 34092 120 3.0 33 0 6 27060 PAGE 104 104 Untreated filter 2 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.0 40 0 1 288 40 3.0 40 0 2 552 60 3.0 38 0 3 612 80 3.0 34 0 4 1368 100 3.0 34 0 5 28644 120 3.0 33 0 6 20724 Experiment 2. Room Temperature: 23 2 C 20 3.0 35 0 1 120 40 3.0 35 0 2 252 60 3.1 35 0 3 648 80 3.2 34 0 4 960 100 3.2 33 0 5 19788 120 3.2 33 0 6 20484 Experiment 3. Room Temperature: 23 2 C 20 3.1 38 1 1 480 40 3.0 33 1 2 816 60 3.0 33 0 3 1260 80 3.0 33 0 4 2412 100 3.0 33 0 5 29232 120 3.0 33 0 6 28572 Experiment 4. Room Temperature: 23 2 C 20 3.0 40 0 1 168 40 3.0 40 0 2 672 60 3.1 38 0 3 1044 80 3.0 36 0 4 2088 100 3.0 33 0 5 30600 120 3.2 33 0 6 35472 Experiment 5 Room Temperature: 23 2 C 20 3.1 40 0 1 384 40 3.1 36 0 2 768 60 3.1 34 0 3 1116 80 3.2 33 2 4 2148 100 3.1 34 0 5 29472 120 3.1 34 0 6 22608 PAGE 105 105 Untreated filter 3 (Room temperature & Low RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.0 53.0 0 1 300 40 3.1 53 0 2 456 60 3.1 52 0 3 636 80 3.2 49 0 4 1296 100 3.2 48 0 5 27684 120 3.1 46 0 6 36816 Experiment 2. Room Temperature: 23 2 C 20 3.1 40 0 1 144 40 3.1 39 0 2 372 60 3.1 39 0 3 780 80 3.1 39 0 4 1392 100 3.2 38 0 5 28068 120 3.1 38 0 6 29412 Experiment 3. Room Temperature: 23 2 C 20 3.1 40 0 1 276 40 3.1 40 1 2 552 60 3.1 37 0 3 588 80 3.1 36 0 4 2112 100 3.2 35 0 5 25200 120 3.1 36 0 6 30348 Experiment 4. Room Temperature: 23 2 C 20 3.1 37 0 1 276 40 3.1 37 0 2 324 60 3.2 35 0 3 480 80 3.1 35 0 4 1284 100 3.1 36 0 5 24924 120 3.1 36 0 6 21528 Experiment 5 Room Temperature: 23 2 C 20 3.1 40 0 1 456 40 3.1 38 1 2 444 60 3.1 36 0 3 744 80 3.2 35 0 4 2136 100 3.2 36 0 5 30924 120 3.2 36 0 6 25944 PAGE 106 106 Iodine-treated filter 1 (Room temperature & High RH) Experiment 1. Room Temperature: 23 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 2.6 100 0 1 300 40 2.7 100 0 2 564 60 2.6 100 0 3 1008 80 2.7 100 0 4 1800 100 2.6 100 0 5 32844 120 2.6 100 0 6 44304 Experiment 2. Room Temperature: 23 2 C 20 2.9 100 0 1 456 40 2.5 99 0 2 684 60 2.4 100 0 3 1176 80 2.7 100 0 4 2076 100 2.8 80 0 5 32712 120 2.7 87 0 6 39900 Iodine-treated filter 2 (Room temperature & High RH) Experiment 1. Room Temperature: 23 2 C 20 2.7 95 0 1 516 40 2.8 90 0 2 636 60 2.6 91 0 3 1236 80 2.6 90 0 4 2544 100 2.8 92 0 5 33804 120 2.8 93 0 6 34056 Experiment 2. Room Temperature: 23 2 C 20 2.8 92 1 1 480 40 2.6 86 0 2 804 60 2.6 91 0 3 1128 80 2.8 92 0 4 2220 100 3.0 96 0 5 32712 120 2.8 93 0 6 42312 PAGE 107 107 Iodine-treated filter 1 (High temperature & High RH) Experiment 1. High Temperature : 40 2 C Baseline Time (min) Pressure drop (in. H2O) Relative Humidity (%) Penetration (CFU) Impactor Stage CFU 20 3.0 100 0 1 420 40 3.5 100 0 2 888 60 3.4 100 0 3 1224 80 3.6 100 0 4 2544 100 3.4 100 0 5 36264 120 3.6 100 0 6 45660 Experiment 2. High Temperature : 40 2 C 20 3.8 92 0 1 660 40 3.4 86 1 2 732 60 3.0 91 0 3 1152 80 2.1 92 0 4 2892 100 2.6 96 0 5 39264 120 2.7 93 0 6 44952 Iodine-treated filter 2 (High temperature & High RH) Experiment 1. High Temperature : 40 2 C 20 2.8 100 0 1 1080 40 2.7 100 0 2 1464 60 2.7 100 0 3 2052 80 2.5 100 0 4 3804 100 2.6 96 0 5 36996 120 2.8 98 0 6 47652 Experiment 2. High Temperature : 40 2 C 20 2.7 98 0 1 684 40 2.7 100 0 2 1188 60 3.0 100 0 3 1728 80 2.7 100 0 4 3708 100 2.8 96 0 5 37140 120 2.8 100 0 6 47652 PAGE 108 108 APPENDIX B PROCEDURES FOR PREPARING PLAQUE ASSAY MEDIA MS2 Media With gentle mixing, 1.0 g trypton e, 0.1 g yeast extract, 0.1 g D-glucose, 0.8 g NaCl, and 0.022 g CaCl2 wre added to a total volume of 100 mL of distilled water in a 250-mL flask. The mixed medium was autoclaved at 121 oC for 30 mins. MS2 Agar Media With gentle mixing, 3.0 g trypton e, 0.3 g yeast extract, 0.3 g D-glucose, 2.4 g NaCl, 0.066 g CaCl2, and 0.3 g of Bacto-agar were added to a to tal volume of 300 mL of distilled water in a 500-mL flask. The mixed agar was autoclaved at 121 oC for 30 mins. 1XPBS dilution tube 1.8 g KH2PO4, 15.2 g K2HPO4, and 85 g NaCl were added to 1L of distilled water to make 10XPBS. 1XPBS was prepared by diluting 10XPBS in distilled wa ter. 9-mL aliquots of 1XPBS were dispensed into 16 150 mm test tubes and autoclaved at 121 oC for 30 min. PAGE 109 109 APPENDIX C RAW DATA OF VI RUS EXPERIMENT Table C-1. All glass impinger results at various environmental conditions Iodine-treated filter 1 (Room temperature & Low RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 38 10 3350 99.70 60 0.3 39 30 1590 98.11 90 0.3 39 80 18650 99.57 120 0.2 38 120 19500 99.49 Experiment 2. Temperature: 23 2 C 30 0.2 40 0 4450 100.00 60 0.3 38 0 10550 100.00 90 0.2 38 300 40000 99.25 120 0.2 37 100 48500 99.80 Experiment 3. Temperature: 23 2 C 30 0.2 40 100 16500 99.39 60 0.2 39 120 15000 99.20 90 0.2 39 110 32000 99.66 120 0.2 38 150 34500 99.57 Experiment 4. Temperature: 23 2 C 30 0.2 39 100 19000 99.47 60 0.2 38 250 32000 99.22 90 0.2 39 220 31000 99.29 120 0.3 37 150 15600 99.04 Experiment 5. Temperature: 23 2 C 30 0.2 39 110 67000 99.84 60 0.2 38 300 61000 99.51 90 0.2 38 170 12600 98.65 120 0.2 37 260 25000 98.96 PAGE 110 110 Iodine-treated filter 2 (Room temperature & Low RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 40 33 14450 99.78 60 0.2 39 8 18350 99.96 90 0.2 39 44 22250 99.80 120 0.2 39 23 24400 99.91 Experiment 2. Temperature: 23 2 C 30 0.2 40 15 59500 99.97 60 0.2 39 0 66000 100.00 90 0.2 39 0 36000 100.00 120 0.2 40 100 49500 99.80 Experiment 3. Temperature: 23 2 C 30 0.2 40 100 17000 99.41 60 0.2 38 0 6500 100.00 90 0.3 38 150 26000 99.42 120 0.3 37 200 29500 99.32 Experiment 4. Temperature: 23 2 C 30 0.2 39 120 56700 99.79 60 0.2 39 400 70000 99.43 90 0.2 38 120 27000 99.56 120 0.2 38 300 20500 98.54 Experiment 5. Temperature: 23 2 C 30 0.2 40 90 29500 99.69 60 0.2 39 130 19000 99.32 90 0.2 40 90 32000 99.72 120 0.2 38 110 60000 99.82 PAGE 111 111 Untreated filter 1 (Room temperature & Low RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 40 2320 21700 89.31 60 0.2 40 2115 25600 91.74 90 0.2 39 2405 21200 88.66 120 0.2 38 2370 28350 91.64 Experiment 2. Temperature: 23 2 C 30 0.2 40 345 7400 95.34 60 0.2 39 550 11650 95.28 90 0.2 38 570 7800 92.69 120 0.2 38 350 7300 95.21 Experiment 3. Temperature: 23 2 C 30 0.2 40 3480 47850 92.73 60 0.2 39 2820 29950 90.58 90 0.2 38 1850 26200 92.94 120 0.2 38 2310 28900 92.01 Experiment 4. Temperature: 23 2 C 30 0.2 39 540 6000 91.00 60 0.2 38 270 3550 92.39 90 0.2 38 185 3050 93.93 120 0.2 38 315 4950 93.64 Experiment 5. Temperature: 23 2 C 30 0.2 39 680 8350 91.86 60 0.2 39 580 7600 92.37 90 0.2 39 455 6200 92.66 120 0.2 38 700 9000 92.22 PAGE 112 112 Untreated filter 2 (Room temperature & Low RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 40 430 4000 89.25 60 0.2 39 280 4250 93.41 90 0.2 39 445 4400 89.89 120 0.2 38 275 3200 91.41 Experiment 2. Temperature: 23 2 C 30 0.2 39 1660 10650 84.41 60 0.2 39 515 9400 94.52 90 0.2 38 1110 10200 89.12 120 0.2 38 490 5350 90.84 Experiment 3. Temperature: 23 2 C 30 0.2 40 915 8450 89.17 60 0.2 39 595 5600 89.38 90 0.2 38 1365 18500 92.62 120 0.2 38 730 9400 92.23 Experiment 4. Temperature: 23 2 C 30 0.2 39 1705 17050 90.00 60 0.2 39 805 8350 90.36 90 0.2 39 865 7800 88.91 120 0.2 38 710 9150 92.24 Experiment 5. Temperature: 23 2 C 30 0.2 40 1480 22300 93.36 60 0.2 39 1220 15050 91.89 90 0.2 40 535 5250 89.81 120 0.2 38 415 4800 91.35 PAGE 113 113 Iodine-treated filter 1 (R oom temperature & Medium RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.5 56 90 17700 99.49 60 0.3 51 40 10700 99.63 90 0.3 50 20 16900 99.88 120 0.3 53 0 8800 100.00 Experiment 2. Temperature: 23 2 C 30 0.3 60 1 5150 99.98 60 0.2 53 0 5100 100.00 90 0.3 51 3 2650 99.89 120 0.3 56 3 4750 99.94 Experiment 3. Temperature: 23 2 C 30 0.3 57 0 2500 100.00 60 0.2 51 65 6950 99.06 90 0.2 51 55 9350 99.41 120 0.2 51 10 7050 99.86 Experiment 4. Temperature: 23 2 C 30 0.3 49 10 2150 99.53 60 0.3 52 20 2300 99.13 90 0.3 60 20 4200 99.52 120 0.3 57 0 4000 100.00 Experiment 5. Temperature: 23 2 C 30 0.2 39 0 3450 100.00 60 0.2 38 0 1100 100.00 90 0.2 38 0 4150 100.00 120 0.2 37 0 2350 100.00 PAGE 114 114 Iodine-treated filter 2 (R oom temperature & Medium RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 58 0 3000 100.00 60 0.3 54 0 6000 100.00 90 0.2 59 0 1850 100.00 120 0.2 56 0 2000 100.00 Experiment 2. Temperature: 23 2 C 30 0.3 56 0 950 100.00 60 0.2 54 0 3650 100.00 90 0.3 57 10 700 98.57 120 0.3 56 10 300 96.67 Experiment 3. Temperature: 23 2 C 30 0.2 57 0 3100 100.00 60 0.3 59 1 2150 99.95 90 0.3 57 0 1400 100.00 120 0.3 59 0 1450 100.00 Experiment 4. Temperature: 23 2 C 30 0.2 55 0 550 100.00 60 0.2 57 0 700 100.00 90 0.2 56 0 1700 100.00 120 0.2 57 0 2050 100.00 Experiment 5. Temperature: 23 2 C 30 0.3 69 0 1750 100.00 60 0.3 59 0 1750 100.00 90 0.3 60 0 2300 100.00 120 0.3 58 0 600 100.00 PAGE 115 115 Untreated filter 1 (Room temperature & Medium RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.3 47 1100 43000 97.44 60 0.3 46 900 20000 95.50 90 0.3 45 1000 19000 94.74 120 0.3 45 1300 21000 93.81 Experiment 2. Temperature: 23 2 C 30 0.3 43 6350 121000 94.75 60 0.2 47 10100 178000 94.32 90 0.3 46 9050 101500 91.08 120 0.3 45 9950 245000 95.94 Experiment 3. Temperature: 23 2 C 30 0.3 46 4150 41000 89.88 60 0.3 45 4300 44000 90.23 90 0.3 46 4150 55000 92.46 120 0.3 46 3150 72000 95.63 Experiment 4. Temperature: 23 2 C 30 0.3 51 1000 14050 92.88 60 0.3 49 685 12400 94.48 90 0.3 47 925 14550 93.64 120 0.3 48 1000 17500 94.29 Experiment 5. Temperature: 23 2 C 30 0.3 50 3050 33500 90.90 60 0.3 46 1800 22500 92.00 90 0.3 45 1600 26500 93.96 120 0.3 44 2900 30000 90.33 PAGE 116 116 Untreated filter 2 (Room temperature & Medium RH) Experiment 1. Temperature: 23 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.2 48 3150 33000 90.46 60 0.2 47 1850 21150 91.25 90 0.2 47 1650 16200 89.82 120 0.2 47 750 16700 95.51 Experiment 2. Temperature: 23 2 C 30 0.2 47 1000 14800 93.24 60 0.2 48 1650 15200 89.15 90 0.2 47 1900 19000 90.00 120 0.2 45 1100 19000 94.21 Experiment 3. Temperature: 23 2 C 30 0.2 49 2650 28500 90.70 60 0.2 47 3600 39000 90.77 90 0.2 47 2650 35500 92.54 120 0.2 45 3350 31500 89.37 Experiment 4. Temperature: 23 2 C 30 0.2 47 1300 16350 92.05 60 0.2 49 1350 16250 91.69 90 0.2 45 1900 13500 85.93 120 0.2 45 1700 24200 92.98 Experiment 5. Temperature: 23 2 C 30 0.2 47 3250 39500 91.77 60 0.2 47 4050 44000 90.80 90 0.2 44 2100 25500 91.77 120 0.2 45 3700 41500 91.08 PAGE 117 117 Iodine-treated filter 1 (High temperature & Low RH) Experiment 1. Temperature: 40 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.3 39 0 800 100.00 60 0.3 38 0 445 100.00 90 0.3 38 0 26500 100.00 120 0.3 40 0 38000 100.00 Experiment 2. Temperature: 40 2 C 30 0.3 40 0 47500 100.00 60 0.3 39 0 43500 100.00 90 0.3 39 0 40500 100.00 120 0.3 38 0 41000 100.00 Experiment 3. Temperature: 40 2 C 30 0.3 39 0 53500 100.00 60 0.3 38 0 50000 100.00 90 0.3 38 0 57500 100.00 120 0.3 38 0 58500 100.00 Experiment 4. Temperature: 40 2 C 30 0.3 39 0 54500 100.00 60 0.3 39 0 62000 100.00 90 0.3 40 0 28500 100.00 120 0.3 38 0 40500 100.00 Experiment 5. Temperature: 40 2 C 30 0.3 39 0 16000 100.00 60 0.3 40 0 19500 100.00 90 0.3 39 0 18500 100.00 120 0.3 39 0 17500 100.00 PAGE 118 118 Iodine-treated filter 2 (High temperature & Low RH) Experiment 1. Temperature: 30 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.3 39 1 8300 99.99 60 0.3 38 0 6150 100.00 90 0.3 39 1 9200 99.99 120 0.3 34 0 11300 100.00 Experiment 2. Temperature: 30 2 C 30 0.3 42 1 5150 99.98 60 0.2 41 0 5100 100.00 90 0.3 37 1 2650 99.96 120 0.3 32 3 4750 99.94 Experiment 3. Temperature: 30 2 C 30 0.2 40 0 20000 100.00 60 0.2 32 1 17000 99.99 90 0.2 36 1 14000 99.99 120 0.2 38 1 21500 99.99 Experiment 4. Temperature: 30 2 C 30 0.3 34 0 2800 100.00 60 0.3 35 0 1550 100.00 90 0.3 35 0 5950 100.00 120 0.3 35 0 4000 100.00 Experiment 5. Temperature: 30 2 C 30 0.3 34 4 3500 99.89 60 0.3 36 1 500 99.80 90 0.3 46 0 2500 100.00 120 0.3 35 1 4450 99.98 PAGE 119 119 Untreated filter 1 (High temperature & Low RH) Experiment 1. Temperature: 30 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.3 33 3550 101000 96.49 60 0.3 35 6900 74500 90.74 90 0.3 40 3950 102500 96.15 120 0.3 39 4850 119000 95.92 Experiment 2. Temperature: 30 2 C 30 0.3 36 2850 87500 96.74 60 0.2 35 3100 137500 97.75 90 0.3 33 6950 50000 86.10 120 0.3 35 8650 90000 90.39 Experiment 3. Temperature: 30 2 C 30 0.3 40 580 14000 95.86 60 0.3 39 1470 18000 91.83 90 0.3 43 1135 14000 91.89 120 0.3 35 825 14500 94.31 Experiment 4. Temperature: 30 2 C 30 0.4 27 2850 155000 98.16 60 0.2 33 5450 90000 93.94 90 0.3 34 4200 134500 96.88 120 0.3 30 5550 35000 84.14 Experiment 5. Temperature: 30 2 C 30 0.2 41 3400 99500 96.58 60 0.2 40 3400 80500 95.78 90 0.2 33 3000 112000 97.32 120 0.2 37 6000 97000 93.80 PAGE 120 120 Untreated filter 2 (High temperature & Low RH) Experiment 1. Temperature: 30 2 C Time (min) Pressure drop (in. H2O) Relative Humidity (%) Experiment (PFU/mL) Control (PFU/mL) Removal Eff. (%) 30 0.3 38 2335 16500 85.85 60 0.3 36 360 27000 98.67 90 0.3 38 1705 16000 89.34 120 0.3 37 2430 35000 93.06 Experiment 2. Temperature: 30 2 C 30 0.3 42 1785 29500 93.95 60 0.3 42 895 33500 97.33 90 0.3 43 1065 31500 96.62 120 0.3 38 2210 19000 88.37 Experiment 3. Temperature: 30 2 C 30 0.3 38 2050 34500 94.06 60 0.2 32 1750 29000 93.97 90 0.3 35 2150 32500 93.38 120 0.3 42 2400 35000 93.14 Experiment 4. Temperature: 30 2 C 30 0.3 34 1200 14000 91.43 60 0.3 38 950 8500 88.82 90 0.3 40 850 12500 93.20 120 0.3 39 950 15000 93.67 Experiment 5. Temperature: 30 2 C 30 0.3 25 3050 36500 91.64 60 0.3 29 2900 28500 89.82 90 0.3 39 3000 15000 80.00 120 0.3 40 2100 11500 81.74 PAGE 121 121 APPENDIX D PARTICLE SIZE DISTRIBUTION OF NUMBER-BASED, MASS-BASED, AND INFECTIOUS VIRUSES Figure D-1. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aeros ols generated from st erile DI water at medium relative humidity. Figure D-2. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aerosols generated from steril e DI water at high relative humidity. PAGE 122 122 Figure D-3. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aeros ols generated from tryptone solution at medium relative humidity. Figure D-4. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aeros ols generated from tryptone solution at high relative humidity. PAGE 123 123 Figure D-5. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aeros ols generated from artificial saliva at medium relative humidity. Figure D-6. Particle size dist ribution by number (solid line), mass (dotted line), and infectious count (mean with error bars) for MS2 aerosol s generated from artif icial saliva at high relative humidity. PAGE 124 124 APPENDIX E RAW DATA OF CHARACTERIZATION EXPERIMENT Table E-1. Bioassay resu lts (plaque-forming units) MS2 aerosols generated from sterile DI water Low RH (25%) Particle diameter (nm) Set 1 Set 2 Set 3 30 2963 1200 4500 60 1028 285 510 90 1238 2700 2980 120 1043 3375 3413 150 2483 4222 4118 200 2558 7050 9375 230 14850 15675 10800 Medium RH (45%) 30 8 353 53 60 98 143 30 90 645 315 120 45 630 240 150 330 135 315 200 585 75 230 135 540 420 High RH (85%) 30 255 53 60 23 540 38 90 45 555 120 180 540 45 150 585 810 270 200 570 1605 225 230 315 2625 180 MS2 aerosols generated from tryptone solution Low RH (25%) Particle diameter (nm) Set 1 Set 2 Set 3 30 120 225 45 PAGE 125 125 60 75 435 75 90 120 990 105 120 300 675 120 150 435 735 60 200 1320 1170 870 230 2520 2355 2220 Medium RH (45%) 30 15 255 45 60 60 300 120 90 120 570 120 120 105 675 690 150 855 1200 1800 200 1215 2400 2220 230 1905 2985 3195 High RH (85%) 30 45 450 210 60 270 300 270 90 330 300 330 120 1005 525 1005 150 1320 750 1320 200 1050 1035 1050 230 1035 1410 1035 MS2 aerosols generated from artificial saliva Low RH (25%) Particle diameter (nm) Set 1 Set 2 30 45 105 60 75 120 90 30 150 120 90 225 150 735 150 200 615 240 230 1125 1485 PAGE 126 126 Medium RH (45%) 30 225 135 60 525 150 90 990 330 120 1215 675 150 1365 660 200 1485 1755 230 2505 1815 High RH (85%) 30 675 495 60 765 885 90 990 975 120 1185 1185 150 1335 1305 200 1815 1800 230 3450 3705 Table E-2. Polymerase chain reaction results ( ng) MS2 aerosols generated from sterile DI water Low RH Medium RH High RH Particle diameter (nm) Set 1 Set 2 Set 1 Set 2 Set 1 Set 2 30 0.60 1.27 0.40 1.22 90 0.29 0.42 120 0.50 1.87 0.60 0.18 0.81 0.40 200 0.63 1.91 0.52 1.72 1.05 1.73 MS2 aerosols generated from tryptone solution 30 0.17 1.18 0.33 2.70 7.39 90 0.67 2.68 120 0.70 2.71 1.19 1.70 0.57 3.28 200 0.65 7.01 0.72 4.28 0.79 4.50 MS2 aerosols generated from artificial saliva 30 0.29 0.25 0.69 0.62 0.63 0.94 120 1.26 1.31 1.40 1.69 1.69 2.27 200 2.38 2.94 2.52 6.20 7.25 8.66 PAGE 127 127 LIST OF REFERENCES ACGIH (2001) Documentation of the threshold limit values and biological exposure indices. Cincinnati, OH: American Conference of Governmental Industrial Hygienists. Adams, M. H. (1948) Surface inactivation of bacterial viruses and of proteins. J Gen Physiol 31, 417-431. Alvarez, A. J., Buttner, M. P. and Stetzenbach, L. D. 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She entered the Ph.D program of the Envir onmental Engineering and Sciences at the University of Florida from fall 2004. She worked with Dr. Chang-Yu Wu, as a research assistant in the Aerosol and Particulate Research La boratory (APRL). Her current study involves characterization of virus aerosols generated from various spray me dia. She received her Ph.D. from the University of Flor ida in the summer of 2009.