Organism
We obtained rhinovirus 16 strain 11757 from ATCC (Manassas, Virginia) with a titer of 107.25 particles per 0.2 ml. The suspension was diluted with phosphate buffered saline (PBS) to create four different concentrations to be used in the experiment.
Aerosolization and chamber description
We generated a rhinoviral aerosol with a 6-jet Collison nebulizer (model CN-38, BGI, Waltham, MA), which generates 2-μm mass-median diameter (MMD) droplets when run at 20 psig of air pressure [31]. The nebulizing solution consists of PBS, diluted virus, and 0.05 g/l uranine. We used the uranine as a fluorescent tracer, so that some filters could be used to determine the percentage of actual collection versus theoretical collection. The aerosol was generated in a specially designed aerosolization chamber that had been used in studies of UV sensitivity of Serratia marcescens and bacilli Calmette-Guérin (Figure 1) [32].
The chamber consists of four parts: a drying jar, a 165 mm square duct where the aerosol first enters the chamber, a UV exposure chamber, and sampling ports. We attached a Collison nebulizer to the drying jar, where the aerosol is given time to dry, before entering the square duct through a 9-port manifold, which creates a uniformly distributed aerosol. The aerosol next enters the UV exposure chamber. The UV exposure chamber is comprised of a fused quartz UV exposure window (279 × 254 mm), on which a UV fixture containing six 36-watt UV lamps (Lumalier, Memphis, Tennessee) sits. We inserted two types of screens beneath the UV fixture to adjust the level of irradiance. Downstream of the exposure chamber a slide contains three ports. One port is used to collect the aerosol sample with our sampling filters (Teflo 2.0 μm pore, Pall Gelman, Ann Arbor, Michigan) mounted in filter cassettes (Millipore Corp., Bedford, Massachusetts) while the other ports can be loaded with two Teflo filters in series, to allow a bypass while changing sampling filters. Two filters were used in the bypass to ensure that no aerosol escaped the chamber. The multiple ports allow changing of filters without interrupting the aerosol flow. Makeup air passes through a Teflon filter prior to entering the chamber to prevent environmental microorganisms from entering the chamber.
Filtered air was pumped through the chamber at a rate of 8.5 l/min monitored by a rotameter. To provide additional protection, the experimental chamber was housed within a Class II, Type A biosafety cabinet. Although temperature and %RH were not controlled, they were measured with continuous monitors (HOBO H08-004-02 four channel data loggers, Onset Computer Corp., Bourne, Massachusetts) downstream of the sampling filters.
Aerosol procedures
Airflow through the chamber was maintained at 8.5 l/min for 10 minutes. We then began the aerosolization of the rhinoviral solution and allowed the aerosol to flow through the bypass filter for 20 minutes. Air samples were then collected for 10 minutes. For sampling runs that included testing for effects of UV exposure, the UV radiation was turned on and allowed to warm up for 10 minutes while airflow was directed through the bypass filter. An air sample was then collected for 10 minutes. Three replicates of UV exposed and nonexposed particle samples were collected during each sampling run. Aerosols were exposed to an irradiance level of 30 mW/m2 for 22.8 s for a UV dose of 684 mJ/m2.
Spiking experiments
Prior to the aerosol experiments, we spiked filters with progressively more dilute viral suspensions to determine the limit of detection of our extraction and semi-nested RT-PCR assay. For spiking, 1 μl methanol was placed on the filter as a wetting agent followed by 1 μl of diluted virus. Filters were and allowed to dry prior to extraction. We also spiked one filter prior to each aerosol chamber run as a positive control. To test the stability of virus on the filters, a test was conducted in which spiked filters were used to collect air samples for one week in our laboratory, which has single pass air and is therefore an unlikely source of rhinovirus.
Filter extraction
We placed the filters face down in a 60 mm petri dish using sterile forceps. We then added 560 μl of prepared AVL buffer containing carrier RNA (Qiagen QIAamp Viral RNA Mini Kit) and 140 μl phosphate buffered saline (PBS). After placing the lids on the dishes, the dishes were secured to an orbital tabletop shaker (VWR Scientific Products, Thorofare, New Jersey) and rotated for 20 minutes at 360 RPM. After shaking, we pipeted the eluted material from the petri dish into a sterile microfuge tube.
RNA extraction
We extracted the RNA from the filter samples with the Qiagen QIAamp Viral RNA Mini Kit following manufacturer's protocol. The use of the Qiagen QIAamp Viral RNA Mini Kit resulted in 80 μl of purified viral RNA.
Semi-nested RT-PCR amplification
Following extraction, we first amplified the picornavirus RNA using primers OL26 (5'-GCA CTT CTG TTT CCC C-3') [33] and OL27 (5'-CGG ACA CCC AAA GTA G-3') [33] using the Invitrogen SuperScript One-Step RT-PCR with PLATINUM Taq Kit (Invitrogen Corp., Carlsbad, California). The PCR mixture contained 2 × reaction buffer (0.4 mM of each dNTP, 2.4 mM MgSO4), 1 μl of each primer (0.2 μM), 1 μl RT/PLATINUM Taq Mix and 10 μl of the RNA template in a final volume of 50 μl. We conducted the PCR reaction in a MJ Research (Watertown, Massachusetts) PTC-200 Peltier Thermal Cycler programmed with an initial cDNA synthesis and pre-denaturation step of 50°C for 30 minutes and 94°C for 2 minutes followed by then 36 cycles of denature, annealing and extension (15 s at 94°C, 30 s at 55°C, 30 s at 68°C).
We then amplified the PCR products from the RT-PCR step using primers OL26 (5'-GCA CTT CTG TTT CCC C-3') and JWA-1b (5'-CAT TCA GGG GCC GGA GGA-3') [34]. The PCR mixture contained 2 × reaction buffer (0.4 mM of each dNTP, 2.4 mM MgSO4), 1 μl of each primer (0.2 μM), 0.25 μl AmpliTaq (Applied Biosystems, Foster City, California) and 10 μl of RT-PCR product in a final volume of 50 μl. We conducted the PCR reaction for 36 cycles of denature, annealing and extension (15 s at 94°C, 30 s at 55°C, 30 s at 68°C), followed by a final extension of 4 min at 72°C.
Gel electrophoresis
Amplified products were detected by electrophoresis analysis on 2% agarose gel containing 8 μl of ethidium bromide per 200 ml of gel followed by examination under UV light.
PCR products
The OL26 and OL27 primer pair generates a 388 base pair amplicon from a region of the 5' noncoding region of the picornavirus genome. The semi-nested primer pair of OL26 and JWA-1b generates an amplicon of ~292 base pairs from within the original amplicon. The 292 bp amplicon does not differentiate between rhinovirus and enterovirus [34].
Uranine analysis
Uranine was used as a fluorescent tracer, which allowed us to determine a percent yield for each sampling run. To determine the amount of aerosol collected on each filter, uranine analysis was conducted on three filters per sampling run, one each from the beginning, middle and end of each run. The filters used for uranine analysis were removed from cassette holders and placed into a 100 mm test tube with sterile forceps. We added 5 ml of PBS to each tube and sonicated for an hour with vortexing every 15 minutes. We also collected samples of the nebulizing fluid before and after nebulization. Filter samples and nebulizing fluid samples were diluted with 0.01 N NaOH and analyzed with a spectrofluorometer (excitation 485 nm and emission 535 nm) (Tecan Corp. Spectrafluor Plus, Mannedorf, Switzerland). We averaged the results of the three filters to calculate an estimated percent yield for the sampling run.
Data analysis
The amount of uranine nebulized during each sampling run was determined by measuring the amount of fluid consumed and by measuring the fluorescence of nebulizing fluid before and after each sampling run. Using the total aerosolization time during the sampling run, we determined the amount of uranine that theoretically would be on each filter if we collected 100% of the aerosol generated during the 10-minute exposure of each filter. Using the filter samples that were analyzed for uranine, we determined the actual amount of uranine collected per sample. We averaged the three filter samples per run and divided by the total amount of uranine consumed to determine a percent yield (observed/expected) for the sampling run.
We computed the expected number of TCID50 on each filter sample from the known TCID50 and fluid volume added to the nebulizer, the amount of nebulizing fluid consumed per minute, and the sampling time. The expected TCID50 was adjusted for the yield for the uranine samples.