Study design

The experimental room has grounded metal walls, with a volume of 19 m3 (B250*L330*H235cm). One active and one inactive ionizer device, designed for collection and analysis of particles in the air, were placed in the room at equal distance (215 cm) from the nebulizer (Aiolos Albatross, Aiolos, Sweden), with a distance of 64 cm between the ionizers. A particle counter (PortaCount Plus, TSI Incorporated, USA) was used before and during the experiment. Before the start of aerosol experiments, the room was emptied on particles by the active ionizer and the collector plate was discarded before the experiments begun and replaced with a new collector plate. The experiments were continued until the particle counts were back to basal level, usually reached within 40 min. Humidity and temperature conditions were measured initial to each aerosol experiments.

Ionizer technology and device

The ionizing device used in this study was developed on the basis of the ion-flow ionizing technology from LightAir AB, Solna, Sweden (www.lightair.com) and was modified for this work by the Department of Microbiology, Karolinska Institute, Stockholm, Sweden. The device (size of 13 × 35 cm) was modified by installing a plastic-cup with a conductive surface of 47 mm in diameter (GP plastindustri, Gislaved, Sweden) as the collector plate (Fig. 1). The collector plate has for safety reasons a very low current, less than 80 μA, however the ionizer accelerates an extremely high voltage of more than 200,000 eV. The ionizer creates electrons, which will render surface molecules of particles in air negatively charged thus attracting them to the positively charged collector plate. This device generates approximately 35 000 billion electrons per second (www.lightair.com) with a steady-state ozone concentration below the detection limit (0,002 ppm) as tested by VTT Technical Research Center of Finland, Tampere, Finland. It has also been ozone tested and certified by ARB (Air Resources Board) in the US. After the end of the sampling period, the ionizer was turned off and the collector plate was covered with a lid and stored at −20 °C until analysis. Viruses captured on the collector plates were analyzed by a RT-qPCR for rotavirus, CaCV and influenza virus and the results from the active- and inactive ionizers were compared. Scanning- and transmission electron microscopy were used for visualization of collected viruses and latex-particles.

Aerosol experiments of virus and latex particles

Different amounts of rhesus rotavirus (genotype G3P[3]), influenza virus (strain H1N1, Salomon Island, inactivated) and CaCV strain 48 (genus Vesivirus) were diluted in water to a final volume of 5 mL. In aerosol experiments for scanning electron microscopy and infectivity analysis, virus was diluted in Eagles MEM. Virus suspensions in different concentrations were distributed as aerosols in the room by the use of a nebulizer. Each experiment was performed in triplicates and collection of aerosolized virus and latex particles were performed during 40 min.

Transmission electron microscopy (TEM)

Carbon/Formvar-coated 400 mesh copper grids were placed on the collector during aerosol experiment with influenza- and rotavirus. Grids were then rehydrated in Eagles MEM containing 1% bovine serum albumin (BSA) before being negatively stained with 2% phosphotungstic acid and analyzed by TEM. Ten grid squares were analyzed per specimen and the number of virus particles per unit area was calculated.

Scanning electron microscopy (SEM)

Collected samples were added on the surface of a polycarbonate 0.6 μm filter (Nucleopore, Inc) which was fitted to an airtight gadget (GP Plastindustri AB, Gislaved, Sweden). The filter was dried in room temperature, coated with 40 Å thick layer of ionized gold and analyzed by SEM (Philips High Resolution SEM 515). The method has previously been used and reported in studies of cytomegalovirus as well as cerebrospinal fluid41,42,43.

Extraction of viral RNA from collector plates

The attached viral particles on the collector plates were lyzed with 1 mL of viral lysis buffer (buffer AVL, QIAamp viral RNA mini kit) added directly into the collector plate and immediately proceeded for extraction of viral RNA using QIAamp Viral RNA Mini Kit (Cat.no: 52906 Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Each sample was eluted with 60 μL of RNase-free water containing 0.04% sodium azide (AVE buffer; Qiagen, Hilden, Germany).

Reversed transcription of extracted viral RNA

28 μL of the extracted viral RNA was mixed with 50 pmol of Pd(N) 6 random hexamer primer (GE-Healthcare, Uppsala, Sweden) and quickly chilled on ice for 2 min, followed by the addition of one Illustra Ready-To-Go reverse transcriptase PCR (RT-PCR) bead (GE-Healthcare, Uppsala, Sweden) and RNase-free water to a final volume of 50 μL. For rotavirus, an initially denaturation step at 97 °C for 5 min was performed to denature the dsRNA. The reverse transcription (RT) reaction was carried out for 40 min at 42 °C to produce the cDNA later used for real-time PCR.

Quantitative real-time PCR for rotavirus

Rhesus rotavirus was detected and quantified using a LUX real-time PCR assay as described previously7. This real-time PCR uses labeled primers with different fluorophores for each VP6 subgroup and external plasmid standards for semi-quantification44.

Quantitative real-time PCR for CaCV

CaCV was detected and quantified using a SYBR green assay on a ABI prism 7500 (Applied Biosystems, Foster City, CA) with primers; (final concentration 200 nM) CaCV-3 (5-ACCAACGGAGGATTGCCATC-3´ (nucleotides 393 to 410 according to GenBank accession no. AF053720) and CaCV-4 (5´-TAGCCGATCCCACAAGAAGACA-3´ (nucleotides 452 to 474), specific for CaCV strain 48. The reaction was performed with 2 μL cDNA in 10 μL 2X SYBR Green PCR Master Mix (Applied Biosystems) and water to a final volume of 20 μL. The following cycling program was used: 95 °C for 10 min followed by 45 cycles of 95 °C for 15 seconds and 60 °C for 1 min. Melting curve analysis was performed immediately after PCR completion, by heating at 95 °C for 15 seconds, followed by cooling to 60 °C for 1 min and subsequent heating to 95 °C at 0.8 °C min−1 with continuous fluorescence recording. Melting temperatures were determined on all samples using the Sequence Detection Software version 1.3.1 (Applied Biosystems) and visualized by plotting the negative derivatives against temperature.

Sampling for infectivity studies with rotavirus and CaCV

To determine whether the ionizing technology has any influence on virus infectivity, rhesus rotavirus and CaCV was aerosolized and collected on active ionizer collector plates, covered with 1 mL of Eagles MEM. Rotavirus (1 × 106 peroxidase forming units) and CaCV (1 × 106 peroxidase forming units) was aerosolized, each in a total volume of 5 mL and collected for 40 min followed by determination of viral infectivity and number of genome copies.

To determine if ionized air per se had an effect on viral infectivity, rhesus rotavirus was aerosolized and captured on a collector plate containing 1 mL of Eagles MEM, without ionization, placed at a distance of 30 cm from the nebulizer.

To determine if electrostatic attraction of the collector plate affected viral infectivity, rotavirus (2 × 105 peroxidase forming units) and CaCV (2 × 105 peroxidase forming units) in 1 ml of Eagles MEM, were added to inactive and active collector plates for 40 min. The plates were subsequently stored at −20 °C until determination of viral infectivity and number of genome copies.

Determination of rotavirus and CaCV infectivity

Rotavirus stock and samples were diluted 1:10 in Eagles MEM and subsequently diluted in two-fold dilutions. Determination of viral infectivity was performed as previously described on confluent Green monkey kidney cells (MA104) in 96-well plates30. CaCV infectivity was determined essentially as for rotavirus with the modification that samples were added to confluent Madin-Darby Canine Kidney (MDCK) cells in 48-well plates and infectivity determined as previously described45 and confirmed by RT-qPCR. To determine the reduction of infectivity, the ratio of viral genome copy numbers versus infectivity was compared between aerosolized virus, virus exposed to active- and inactive collector plates and the viral stock.

Animals

Guinea pigs, strain Hartley, female, 300–350 g, were housed at Astrid Fagraeus Laboratory, Karolinska Institute, according to approved guidelines from the Board of Agriculture and the Council of Europes Convention on vertebrate animals used for scientific purpose. The experimental protocol was approved by the Animal Ethics Committee in Stockholm (Permit Number: N177/11).

Airborne transmission of influenza virus

We use a guinea pig animal model to investigate whether the ionizing technique could prevent transmission of influenza virus infection, since this model have successfully been used as a model of aerosol transmission studies of influenza virus31,33. Human influenza A virus, strain Pan/99 (kindly provided by Peter Palese, New York, USA) was used since this strain has been shown to effectively replicates in the upper respiratory airways and effectively transmit by aerosols but not by fomites in guinea pigs. Female guinea pigs, 300–350 g, strain Hartley, were housed at Astrid Fagreaus Laboratorium, Solna, Stockholm (Ethical permission N177/11). Four animals were anesthetized by an intra peritoneal injection of ketamin (Ketalar el Ketaminol) 50 mg/kg and xylazin (Rompun) 5 mg/kg and infected intranasally with 5 × 103 pfu of Pan/99 virus in 100 uL (50 uL in each nostril). All four infected animals were placed into the experimental cage (Fig. 3, cage “A”). At 30 h p.i., four naïve uninfected guinea pigs were placed next to the transmission cage (Fig. 3, cage “B”) at a distance of 15 cm. Air flowed freely between cages, but direct contact between inoculated and exposed animals was prohibited.

The four naïve guinea pigs were exposed for 24 hours and then put into separately individually ventilated cages, to ensure that no aerosol transmission occurred between the animals. Two identical experiments were performed, with an active and inactive ionizer. The nasally infected animals were removed after the exposure time and lung and trachea biopsies were collected (54 h p.i.) and investigated for influenza virus by RT-qPCR. At 21 days post exposure, serum was collected from the uninfected exposed guinea pigs and the prevalence of antibodies against influenza A virus was determined by ELISA. Sera taken before exposure to the infected guinea pigs (pre-sera) and 21 days days after exposure (post-sera) were analyzed from each animal.

ELISA detection of influenza A antibodies

Briefly, 96-well plates (Nunc, 96 F MAXISORP, Roskilde, Denmark) where coated with formalin-inactivated Influenza A virus H1N1 (SBL Influenza Vaccine, Sanoil Pasteur, Lyon, France) diluted in coating buffer (0.05 M sodium carbonate buffer, pH 9.5–9.7) at 5 μg/mL and incubated at +4 °C over night. Wells were washed x3 (0.9% NaCl and 0.05% Tween-20) and blocked with 3% BSA in PBS buffer for 1 hour at 37 °C. Serum samples were diluted 1:100 and further in two-fold dilutions in dilution buffer (PBS containing 0.5% BSA and 0.05% Tween-20) and incubated for 90 min at 37 °C. Plates were then washed x5 and incubated for 60 min at 37 °C with secondary biotinylated goat-anti guinea pig antibody (Vector, BA-7000) and horseradish-peroxidase (HRP) conjugated Streptavidin (DAKO, Denmark, P0397), both at a dilution of 1:3000. Plates were then washed x5 and 100 μL of tetramethyl benzidine (TMB) substrate (Sigma Aldrich, T-0440-16) was added to each well, the reaction developed for 10 min and stopped by addition of 100 μL of 2 M H 2 SO 4 . Absorbance was measured at 450 nm in an ELISA reader (VersaMax, Molecular Devices). Cut off values were calculated as the average value of negative controls OD and 2 times the SD.

Extraction of influenza RNA from guniea pig tissue

RNA was extracted from trachea and lung tissue of infected guinea pig. Briefly, 100–250 mg of tissue were homogenized with a tissue homogenizer and total RNA extracted with RNAeasy Midi Kit (Qiagen) according to the manufacturer’s instructions.

Quantitative real-time PCR for influenza virus

To detect and quantify influenza A virus on the collector plates as well as in guinea pig tissue samples, we used a One-Step Taq Man real-time RT-PCR assay46 with primers F1-mxA (150 nM) (5´-AAGACCAATYCTGTCACCTCTGA-3´), F3-mxA (150 nM) (5´-CAAGACCAATCTTGTCACCTCT GAC-3´) and R1-mxA (900 nM) (5´-TCCTCGCTCACTGGGCA - 3´) and probes P1-Mx (110 nM) (5´-FAM-TTGTGTTCACGCTCACC–MGB–3´) and P2-Mx (110 nM) (5´-FAM-TTTGTATTCACGCTCACCG–MGB -3´), with the Rotor-Gene Probe RT-PCR Kit (Qiagen). The real-time PCR reaction was performed in a Corbett Rotor-Gene 6000 (Qiagen) with the following cycling protocol: 50 °C for 10 min, followed by 45 cycles of 95 °C for 5 seconds and 57 °C for 15 seconds.