Evidence of Infection among Ferrets exposed to high levels of nebulized NC99.

a<p>For exposures to ferrets 2-C and 2-D, the diffusion dryer was inserted into the line exiting the Collison generator, reducing the humidity at the end of the hour nebulization.</p>b<p>Calculation of inhaled virus based on measured viral RNA in aerosol inhaled during 60 min of aerosol exposure.</p>c<p>Nasal wash was collected on day 2 post exposure (pe) in Exp 1, and daily in Exp 2; culture results are reported for the entire collection as FFU/mL; viral RNA is the peak titer on day 3 pe and is reported as GEq/collection.</p

Comparison of impinger and PTFE filter efficiencies for culturable virus and viral RNA.

a<p>Virus NC99 was nebulized in 6 experiments with impinger alone and 9 experiments with impinger and PTFE filter collections in parallel. The mean (range) total dose nebulized was 1.8 (1.1–2.2)×10⁷ FFU, calculated by virus concentration in Collison at beginning of aerosol generation times fluid volume nebulized for each run.</p>b<p>Impinger collection measured for 5.4 (range 5.3–5.5) L/min, reported as total FFU collected during interval of nebulization. N = 5 at each time point as one outlier value was removed from each group.</p>c<p>PTFE filter collection combines two filters in parallel for total flow of 4.0 L/min, reported as total FFU and total genome equivalents (GEq) of RNA measured by T5000 assay.</p>d<p>Ratio of group means of FFU and GEq RNA collected respectively.</p

Characteristics of the aerosol exposure system.

<p><b>a.</b> Photograph of exposure apparatus during nebulization with Collison generator inside the origin chamber shows the visible cloud of airborne particles in both the left (origin) and right (recipient) chambers. Placement of the Collison generator outside of the origin chamber, connected with 20 cm tubing, resulted in no visible suspended airborne particles in the chambers, and all subsequent experiments reported here had this configuration. The Grimm particle spectrometer is placed on top of the left chamber and the sampling port is located on top of the tunnel. <b>b.</b> The number particle size distribution with GRIMM optical counter. The fitted bimodal distribution has the median diameters of 0.44 and 1.70 µm, and geometric standard deviation of 1.25 and 1.46, for the two size modes, respectively. <b>c.</b> Particle number concentrations detected by Grimm laser-based particle counter sampled during four 30–60 sec intervals of the continuous one-hour nebulization.</p

Comparison of sensitivity of the T5000 and RT-PCR Assays.

<p>Comparison of sensitivity of the assays for samples expected to contain relatively higher concentrations (3.0 to 7.0 log₁₀ Geq) of viral RNA (throat swab and nasal wash) and relatively lower concentrations (1.0 to 4.0 log₁₀ Geq) of viral RNA. The Ibis T5000 Influenza assay was not significantly more sensitive for detecting viral RNA in the airway specimens under the conditions of this experimental infection, but was significantly more sensitive in detection viral RNA in filter and impinger aerosol collection devices (Chi-Squared test, p<0.01).</p

Evidence of viral replication in ferrets exposed to low doses of aerosolized NC99.

<p>a. Actual dose calculated was within 30% of target dose.</p><p>b. Calculations based on viral RNA collected on PTFE filter during 60 min of aerosol exposure.</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

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

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

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