Particle Concentration variation during chamber exposure.

<p>Airborne particles were measured continuously by Grimm spectrometer and recorded as particles per liter of air in this typical exposure. Particle numbers per second vary over two orders of magnitude.</p

Distribution of particle dimensions delivered to recipient chamber.

<p>Particle diameter measured by laser light scattering plotted as log₁₀ particles per liter of air divided into 24 diameter cohorts ranging from 0.25 microns to 12.5 microns. Particles sampled during 10 min. intervals with sampling airflow at 1.0 Lt/min and with one resting ferret infected with Cal/04 virus in donor chamber. Top graph: In side-by-side chamber sampling collected during the middle (green) and end (blue) of the same exposure period, and during an interval of no directed airflow when vacuum is off (control, red) particle numbers of all size cohorts decreased more than 100-fold. Bottom graph: In tunnel exposure chamber samples collected during first hour (blue), second hour (red) and third hour (green) of continuous 3 hour exposure.</p

Histologic grades of inflammation in respiratory tract tissues of ferrets infected with three pandemic H1N1 viruses.

<p>Histologic grades of inflammation in respiratory tract tissues of ferrets infected with three pandemic H1N1 viruses.</p

Aerosol transmission is more efficient after 20 h- than 3 h- exposures.

<p>Viral RNA (log₁₀ genome equivalents (GEq) in total sample) in throat swabs from four donors (Do, red) infected intranasally 24 hours previously with Cal/04, and in four recipients (R, blue) each exposed to exhaled aerosols of one of the donors for either 3 hours (A and B) or 20 hours (C and D).</p

Exhaled viral RNA from donors infected with either NC/99 (A and B) or Cal/04 (C and D) virus.

<p>RNA was measured in each filter sample by two RT-qPCR-based assays, detecting a single genomic RNA segment (LRRI assay, solid bar) or six segments (Ibis T5000 assay, checkered bar), and expressed as genome equivalents/1-hour filter collection. Donors were infected intranasally with 10⁶ FFU of either virus 24 hours prior to recipient exposure at a time when all donor nasal washes contained 10⁴–10⁵ FFU/mL. Each donor exposed three recipient ferrets and each of their corresponding collections (F1, F2, and F3) were the mean RNA levels from three filters each collecting airborne particles for 1 hour during the 3-hour exposure period.</p

Efficiency of transmission success is inversely correlated with level of illness in the ferret aerosol-donor. A

<p>. Comparison of percent transmissions detected by positive culture or positive RT-qPCR of nasal washes or throat swabs of recipients tested 1−3 days post-exposure, for each strain of pandemic H1N1 2009 influenza A virus. Lack of transmission of NC/99 is shown as left-hand column for comparison. <b>B</b>. Group mean weight change in the donors following intranasal infection with the three pandemic H1N1 strains. <b>C</b>. Photographs of whole lung tissue at necropsy (day 5) of donor ferrets infected with Cal/04 (C2, middle photo) or Cal/07 (C1 and C3). Cal/07-infected lungs display multiple regions of firm, dusky tissue representing pneumonitis confirmed by histology, not seen in Cal/04-infected lungs.</p

Aerosol Transmission from donors infected by aerosolized virus.

<p>Transmission of Cal/04 between donors infected by nebulized virus and recipients exposed continuously for 4 days. <b>A</b>. Serial viral RNA titers from throat swabs of donors and recipients, and samples of aerosols collected during continuous exposures, measured by single-target RT-qPCR. <b>B</b>. Viral RNA captured on PTFE filters during a 1 hour interval each day after donor ferret infection, and measured by T5000 assay.</p

Comparison of Transmission efficiency of pandemic 2009 H1N1 strains during early and later phases of donor infection.

<p>Single donor-single recipient exposures in which the interval between donor instillation and recipient exposure was varied from 1, 3, or 5 donor dpi. Viral culture (hatched bar) is expressed as FFU/mL (total sample) of nasal wash collected on day of exposure (donors) or the greatest value found on day 1−3 post-exposure (recipients). Aerosol data are expressed as genome equivalents/1-hour filter collection; each of three filters was measured in triplicate, and the mean of the three filters reported. For each virus: Cal/04 (green), Cal/07 (blue) and Mex/4482 (red), the donor-recipient pair are aligned horizontally across the figure.</p

Exposure chambers. A

<p>. Photograph of tunnel exposure chamber occupied by two sleeping ferrets in the left donor chamber and two active ferrets in the right ‘recipient’ chamber. Note that experiments are described with two ferrets in the donor chamber but no experiments were performed with two ferrets in the recipient chamber. A line diagram of this exposure chamber appears in reference 32. Air is drawn through HEPA filters in the left wall of the donor chamber (left side of photograph) passes through the tunnel where particle size and PTFE filter ports are located, and is withdrawn through the recipient chamber (right side) and exits through two ports into HEPA filters (not visible in photograph). <b>B</b>. Exploded view of exposure chamber without tunnel between donor and recipient ferret, designed to more closely approximate conditions of side-by-side cage exposures in published ferret-model aerosol transmission studies.</p

Viral loads (RNA genome equivalents per mL nasal wash or per g lung tissue) for each donor ferret on dpi 1 or dpi 3.

<p>Necropsy of donor was performed 2 h after exposing recipient ferrets. Data combined from 3 experiments indicated by circles, triangles and diamonds. Lung tissue viral RNA levels for Cal/04 were mean (SD) = 5.94 (2.72) compared to Cal/07+Mex/4482 lung RNA mean (SD) = 8.84 (1.42), significant by t test at p = 0.007. Aerosol transmission success for each virus is exhibited in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033118#pone-0033118-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033118#pone-0033118-g007" target="_blank">7</a>. The NC/99-infected donors did not have viral RNA in lung tissue nor transmitted infection by exhaled aerosol.</p