Statistical analysis of summed clinical signs for each day in ferrets re-challenged with A/Cal.

<p>The group that previously experienced A/Cal+300 ug 244 DI virus (▪) is compared with the group that previously experienced only saline (▴). The p value was determined using a one tailed Mann-Whitney U test.</p

Summary of 244 DI virus-mediated protection of ferrets from influenza caused by A/Cal.

*<p> <b>Statistically significant at ≥95% confidence limits; data presented elsewhere in this report.</b></p><p> <b>ND, not done.</b></p

Summary of A/Cal infectivity in nasal washes.

<p>Panel (a) shows ferrets infected with A/Cal on day 0 and treated with 300 µg 244 DI virus (▪) or infected and treated with 300 µg inactivated 244 DI virus (▴); another group was not infected but treated with 300 µg of active 244 DI virus (•). A standard preparation of A/Cal virus was used to normalise titrations carried out on different days. These varied by less than 4-fold. The dotted line is the limit of sensitivity of the assay (1.92 log₁₀ FFU/ml). Significant reduction in infectivity (by a two-tailed Mann-Whitney U test) in ferrets treated with 244 DI virus is denoted by **. Panels (b) and (c) show details of the statistical analysis on day 2 and 3, respectively.</p

Changes in weight of ferrets over the course of infection with A/Cal.

<p>Shown is the mean group body weight changes in A/Cal influenza virus-infected ferrets treated with (a) 300 µg 244 DI virus (▪) or inactivated 244 DI virus (▴), (b) 30 µg 244 DI virus (▪) or inactivated 244 DI virus (▴). (c) Shows the weight changes in ferrets inoculated with saline (○) or treated with 300 µg of active 244 DI virus (•). Data are expressed as a percentage change compared to the group average weight at day 0. The statistical significance of body weight changes on any one day was determined by a one tailed unpaired t-test and is indicated by an asterisk (p≤0.05).</p

244 DI virus (244) RNA is amplified in nasal washes by A/Cal.

<p>Ferrets were infected with A/Cal on day 0 and treated with 244 DI virus or inactivated 244 DI virus. Levels of 244 DI RNA were determined by quantitative RT-PCR. Mean 244 RNA copy numbers for each ferret group (n = 5) are plotted. Panel (a) shows ferrets that were infected with A/Cal influenza virus and treated with 300 µg 244 DI virus (▪), or 30 µg 244 DI virus (▴), or 300 µg (i) inactivated 244 DI virus (▾), or 30 µg (i) inactivated 244 DI virus (⧫). Panel (b) shows non-infected ferrets that were given 300 µg 244 DI virus (□), or diluent (▵). The dotted line shows the limit of detection. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049394#pone.0049394.s001" target="_blank">Figure S1</a> gives details for individual animals.</p

Effect of virus dose on parameters of infection with influenza A/California/04/09.

1<p>Mean clinical score is calculated as described in Materials and Methods, and expressed as mean score per ferret per day ± standard error of the mean.</p>2<p>Day of onset refers to median onset of respiratory signs and inactivity from four studies.</p><p>ND, not done.</p

Viral RNA loads in ferret respiratory tract tissues.

<p>Ferrets were infected intra-nasally with 10⁶ or 10² pfu Cal/04 and, where indicated, treated with oseltamivir from 6 hr post-infection. Circles show RNA loads for individual animals. Horizontal lines show group means. Filled circles, no treatment; open circles, oseltamivir treated. A, nasal turbinate; B, trachea; C, lung. High, 10⁶ pfu inoculum; Low, 10² pfu inoculum. Samples were taken from 2 ferrets on days 1 to 4, and 3 ferrets on day 5. The sensitivity of the assay was approximately 10³ copies/mg.</p

Effect of decreasing infectious dose on virus shedding.

<p>Ferrets were infected intra-nasally, and nasal washes were collected at the intervals shown for virus plaque assay. Markers show geometric mean nasal wash titre from groups of 5 or 8 ferrets; error bars show standard deviation. For days 10 and 14 post-infection, 2 ferrets per group were used. • (red) high dose (10⁶ pfu), ▪ (green) medium dose (104 pfu), ▴ (blue) low dose (102 pfu) inoculum. The lower limit of detection was 10 pfu/ml.</p

Microscopic changes in ferret nasal cavity and lung, with and without oseltamivir treatment.

<p>A. Nasal cavity, low dose, untreated group, 4 dpi. Propria mucosa is infiltrated by marked numbers of mixed inflammatory cells. Overlying respiratory epithelium comprises attenuated, pre-ciliated, regenerating cells (arrow). B. Nasal cavity, low dose with oseltamivir-treated group, 4 dpi. A mild, mononuclear cell infiltrate within the nasal propria mucosa underlying a normal, pseudostratified, columnar, ciliated epithelium (arrow). C. Lung, low dose, untreated group, 5 dpi. Multifocally extensive, mononuclear cell infiltration of parenchyma. Inset: focal, bronchial gland necrosis (asterisk). D. Lung, high dose, untreated group, 5 dpi. Marked infiltration of parenchyma with inflammatory cells by peribronchial and perivascular oedema (Oe) and bronchial gland necrosis (arrow). E. Lung, low dose, oseltamivir-treated group, 5 dpi. Minimal, parenchymal, mononuclear cell infiltrate (arrow). F. Lung, high dose, oseltamivir-treated group, 5 dpi. Mild, peribronchiolar, mononuclear cell infiltration (arrow). Haematoxylin and eosin.</p

Nasal wash virus titres in the presence or absence of treatment with oseltamivir.

<p>A, prophylactic oseltamivir regimen from 2; B, therapeutic oseltamivir regimen from 6 hr post-infection. • (red) high dose (10⁶ pfu); ▴ (blue) low dose (10² pfu); open symbols represent oseltamivir-treated animals. Means from 5 ferrets (A) or 3–9 ferrets per group (B).</p