Highly efficient collection of infectious pandemic Influenza H1N1 virus (2009) through laminar-flow water based condensation

(SUPPLEMENTAL INFORMATION)

John Lednicky1, Maohua Pan2, Julia Loeb1, Hsin Hsieh3, Arantzazu Eiguren-Fernandez4, Susanne Hering4, Z. Hugh Fan5,Chang-Yu Wu2

1Department of Environmental and Global Health, University of Florida, Gainesville, FL, USA 2Department of Environmental Engineering Sciences, University of Florida, Gainesville, FL, USA 3Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA

4Aerosol Dynamics Inc., Berkeley, CA, USA

5Department of Mechanical and Aerospace Engineering, University of Florida, Gainesville, FL, USA

Figure S1. Physical collection efficiency as a function of particle size, from 6 nm to 10 µm

Figure S2. Schematic diagram of the testing system. The dash line of the BioSampler and the Optical Particle Counter represents tests that were conducted sequentially from the same port of the system as the VIVAS.

Cell Culture

Madin Darby canine kidney cells (MDCK cells, CCL-34) were obtained from the American Type Culture Collection (Manassas, VA, USA) and were propagated as monolayers at 37 °C and 5% CO2 in Advanced Dulbecco’s Modified Eagle’s Medium (aDMEM) (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 2 mM L-Alanyl-L-Glutamine (GlutaMAX, Invitrogen Corp.), antibiotics (PSN; 50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin (Invitrogen Corp.), and 10% (v/v) low IgG, heat-inactivated gamma-irradiated fetal bovine serum (HyClone, Logan, Utah).Prior to use in experiments, the MDCK cells were treated for 3 weeks with plasmocin and verified free of mycoplasma DNA by PCR.

Influenza virus

Influenza virus strain A/Mexico/4108/2009 (pH1N1) is a wild-type H1N1 pandemic 2009 strain that was kindly provided by Drs. Gary Heil (University of Florida) and Gregory Gray (Duke University). The virus was obtained as a low-passage stock and propagated in MDCK cells in serum-free aDMEM plus L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated mycoplasma- and extraneous virus-free trypsin (Worthington Biochemical Company, Lakewood, NJ) in 5% CO2 at 33 °C. The TPCK-trypsin was used at a final concentration of 2 μg/mL. Virus identity was confirmed before use by nucleotide sequencing of the strain-specific hemagglutinin and neuraminidase genes.

VIVAS Physical Collection Efficiency

Collection efficiencies for inert particles were measured for deposition onto water using the 32-jets of the VIVAS as shown in Figure S3. The tested system used the same tube and jet configuration as the present study, but deposition was onto 5 mL of liquid held in a 47 mm Petri dish instead of the 1.5 mL into a 25 mm petri dish of this study. The system was challenged with laboratory generated aerosol, and the deposition efficiency was inferred by comparison of the concentration of particles that penetrated the collection medium, to those measured upstream. To minimize downstream transport losses, the VIVAS system was equipped with a straight, 10 mm ID outlet tube. Comparisons were made for operation with the VIVAS at temperature and with wetted wicks, and with no heating or cooling. Collection efficiency in the size range from 6 nm to 1000 nm were made using atomized ammonium sulfate that was size selected using a differential mobility analyzer, with upstream and downstream particle concentrations measured by a water-based condensation particle counter (TSI-3785). Collection at particle sizes above 800 nm measurements, and as high as 10 µm were made using Arizona Road Dust generated from a fluidized bed, accompanied by upstream and downstream measurements with an aerodynamic particle sizer (TSI Model 3381). Combined data illustrated in Figure S3. There was a slight inconsistency between the Road Dust and sulfate measurements that was not resolved. Nonetheless, the data show that the collection efficiency is >95% for particle sizes from 10 nm to 10 µm.

Figure S3. Photograph of the condensation Particle Collector with its 32 jets impinging onto water held in a Petri dish.