Comparison of viral infection effects on baseline V˙M among Ctrl, HK483, and HK486 mice.

<p><math><msub><mrow><mrow>V</mrow><mo>˙</mo></mrow><mrow>M</mrow></msub></math> was daily measured in the individual mouse at 2 and 1 days before and 1, 2, and 3 days after intranasal inoculation (Day -2, -1, 1, 2, and 3). <math><msub><mrow><mrow>V</mrow><mo>˙</mo></mrow><mrow>M</mrow></msub></math>, minute ventilation; f_R, respiratory frequency; and V_T, tidal volume. Mean ± SE. n = 6, 15, and 7 for Ctrl, HK483 and HK486 mice. * P < 0.05 compared to the data obtained in previous day(s) and † P < 0.05 compared to Ctrl and HK486 mice at the same time-point. Tachypnea (elevation of minute ventilation) is induced in HK483 but not HK486 mice at 2 and 3 dpi.</p

Comparison of viral infection effects on HVR (A) and HCVR (B) among Ctrl, HK483, and HK486 mice.

<p>HVR and HCVR were measured daily in individual mouse at 2 and 1 days before and 1, 2, and 3 days after intranasal inoculation (Day -2, -1, 1, 2, and 3). <math><msub><mrow><mrow>V</mrow><mo>˙</mo></mrow><mrow>M</mrow></msub></math>, minute ventilation; f_R, respiratory frequency; and V_T, tidal volume. Mean ± SE. n = 6, 15, and 7 for Ctrl, HK483 and HK486 mice. * P < 0.05 compared to the data obtained in previous day(s) and † P < 0.05 compared to Ctrl and HK486 mice at the same time-point. HK483 rather than HK486 viral infection produces HVR and HCVR depression at 2 and 3 dpi.</p

Expression of nucleoprotein-immunoreactivity (NP-IR, orange) in substance P immunoreactivity (SP-IR) neurons (green) in the nodose ganglion and tyrosine hydroxylase immunoreactivity (TH-IR) neurons (cyan) in the carotid body.

<p>A: At 2 dpi, nodose ganglion NP-IR appears in a HK483 (right) but not HK486 mice (left) and HK483 virus increases the density of SP-IR in the nodose ganglionic neurons. In contrast, NP-IR is undetectable in the nodose ganglion of infected HK483 and HK486 mice at 1 dpi (data not shown). B: The density of SP-IR in the nodose ganglion is not significantly changed by HK483 virus until 2 dpi. Because of the similarity of the SP-IR data at 2 and 3 dpi (N = 3 and 2, respectively) in either HK483 (64 ± 11 vs. 69 ± 15) and HK486 mice (40 ± 9 vs. 41 ± 11), they were grouped. C: No viral replication in the carotid body of a HK483 mouse in which glomus cells are marked by TH-IR (cyan) at 3 dpi in a HK483 mice. N = 5 for each group; * and † P < 0.05 compared HK483 data to Ctrl and HK486 data respectively.</p

The time to death after viral infection and its correlation with the severity of HVR/HCVR depression.

<p>A: Mortality of Ctrl, HK483, and HK486 mice over the infection period (up to 9 dpi). B and C: HVR and HCVR of individual HK483 mice were plotted against their corresponding death day (at 6, 7, and 8 dpi). Mean ± SE. A: n = 6, 15, and 7 for Ctrl, HK483 and HK486 mice, respectively. B and C: n = 15 HK483 mice. HK483 but not HK486 viral infection led to death at 6–8 dpi and the mice with the larger decrement in HVR or HCVR died earlier.</p

Effects of H5N1 viral infection on animal body temperature (BT), body weight (BW), and metabolism (V˙CO2).

<p>In each mouse of the three groups (n = 6, 15, and 7 for Ctrl, HK483 and HK486 mice), the same measures (BT, BW, <math><msub><mrow>V<mo>˙</mo></mrow><mrow><msub>CO<mn>2</mn></msub></mrow></msub></math>) were repeated at 2 and 1 days before and 1, 2, and 3 days after intranasal inoculation (Day -2, -1, 1, 2, and 3). Mean ± SE. As the results show, there is no significant difference of BT, BW and <math><msub><mrow>V<mo>˙</mo></mrow><mrow><msub>CO<mn>2</mn></msub></mrow></msub></math> among the three groups.</p

Correlation of HVR and HCVR values (V˙M) in HK483 mice.

<p>The values of HRV measured at -2, -1, 1, 2, and 3 days after intranasal inoculation were plotted against those of HCVR at the same times. n = 15 HK483 mice for each day (total 5 days, 75 data points). The significant correlation suggests that a poor HVR is often associated with an inadequate HCVR in HK483 mice.</p

Effects of H5N1 viral infection on the variation of the baseline respiratory intervals (RR).

<p>RR was daily measured in the individual Ctrl, HK483 and HK486 mouse at 2 and 1 days before and 1, 2, and 3 days after intranasal inoculation (Day -2, -1, 1, 2, and 3). A: Poincare plots of RR in an HK486 and an HK483 mice before inoculation (Day -1) and at 1 and 3 days after intranasal inoculation (Day 1, 3). The area of the ellipse describes the distribution of the points with the width of the variation perpendicular to (SD1) and along the line of identify (SD2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147522#pone.0147522.ref027" target="_blank">27</a>]. B: Group data of the variability of baseline respiratory intervals (2500 intervals) in each group. Mean ± SE. n = 6, 15, and 7 for Ctrl, HK483 and HK486 mice. * P < 0.01 compared to the previous time-points; † P < 0.01, compared to Ctrl and HK486 at the given day. A less RR variation is observed after HK483 but not HK486 viral infection at 2 and 3 dpi.</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

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

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