Replication kinetics of rNY1682-WT and rNY1682-D701N <i>in</i><i>vitro</i>.

<p>(<b>A</b>) Confluent monolayers of Madin-Darby canine kidney (MDCK) cells were inoculated with the rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>) viruses at 0.01 TCID₅₀/cell. Supernatants were collected at 2, 24, 48 and 72 hours post inoculation (hpi). (<b>B</b>) Confluent mouse rectal epithelial carcinoma cells (CMT-93) were inoculated with rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>) viruses at 0.01 TCID₅₀/cell and supernatants were harvested at 12, 24, 48, 72 and 96 hpi. (<b>C</b>, <b>D</b>, <b>E</b>) Confluent human lung epithelial cells (Calu-3) were inoculated with rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>) viruses at 0.02 TCID₅₀/cell and supernatants were harvested at 2, 24, 48, 72 and 96 hpi. Cells were incubated at 33^oC (<b>C</b>), 37^oC (<b>D</b>), or 39^oC (<b>E</b>) for the respective growth curves. The averages of triplicate experiments are shown with error bars (+/-) representing the standard deviation, and * indicates that the differences were statistically significant (p<0.001, ANOVA).</p

Pathogenicity of rNY1682-WT, rNY1682-E158G, rNY1682-E627K, and rNY1682-D701N viruses in mice.

<p>Six-week-old female BALB/cJ mice (n=5/group) were inoculated intranasally with 50 µl containing 10⁵ TCID₅₀ of different recombinant viruses (rNY1682-WT (<b>WT</b>), rNY1682-E158G (<b>E158G</b>), rNY1682-E627K (<b>E627K</b>), or rNY1682-D701N (<b>D701N</b>)) or they were mock inoculated (<b>Mock</b>). (<b>A</b>) Morbidity was assessed by weight loss over a 14 day period and is graphed as a percentage of the animals weights on the day of inoculation (day 0). The average body weight of each group is shown with error bars representing SD (+/-), and a significant difference between D701N and WT or E627K was evident as early as 4 d.p.i (*, p<0.01, ANOVA). (<b>B</b>) Mortality associated with infection by the recombinant viruses (rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>)) was also examined. Five-week-old female BALB/cJ mice (n=5/group) were inoculated intranasally with 50 µl containing 1×10³, 10⁴, 10⁵, or 10⁶ TCID₅₀ of rNY1682-D701N or they were mock inoculated. (<b>C</b>) Morbidity was assessed by weight changes over a 14 day period and it is graphed as a percentage of the weight on the day of inoculation (day 0). The average body weight of each group is shown with error bars representing SD. (<b>D</b>) Mortality of the mice was also monitored for 14 days post inoculation. In accordance with our animal welfare protocol, mice that lost more than 25% of their original weight were euthanized for humane reasons.</p

Analysis of viral replication efficiency in the respiratory tracts of mice.

<p>Six-week-old female BALB/cJ mice (n=3/group/time-point) were inoculated intranasally with 50 µl containing 10³ TCID₅₀ of rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>). Animals were euthanized at 12, 24, 48, 96 hpi. The entire lung of each animal was homogenized in 1 ml of media and clarified by centrifugation, and nasal washes were collected from each mouse in 1 ml of media. (<b>A</b>) Viral titers of clarified lung homogenates or (<b>B</b>) nasal washes were determined by TCID₅₀ assay using MDCK cells. The average of each group is shown with error bars representing SD(+/-), and * indicates a statistical difference between WT and D701N (p<0.05, ANOVA). The dotted lines at 1.5 log₁₀ indicate the lower limit of detection of infectious virus.</p

Respiratory droplet transmissibility of rNY1682-WT and rNY1682-D701N viruses in ferrets.

<p>Three ferrets were inoculated with 10⁶ PFU of (<b>A</b>) rNY1682-WT or (<b>B</b>) rNY1682-D701N virus. All ferrets were housed individually in specialized cages that permit exchange of respiratory droplets, but prevent direct or indirect contact between inoculated-contact animal pairs. A naïve ferret was placed in an adjacent cage to an inoculated ferret 1 day post-inoculation (0 day post-contact). Viral replication was determined by titration of nasal washes collected on alternate days from inoculated (left bars) and contact (right bars) ferrets. Values for individual ferrets are shown and the dotted lines at 1 log₁₀ indicate the lower limit of detection of infectious virus.</p

Analysis of cytokine response and virus replication in primary human alveolar epithelial cells.

<p>Human alveolar type I-like cells were infected at 37°C with MOI of 0.01 PFU/cell of the rNY1682-WT (<b>WT</b>) or rNY1682-D701N (<b>D701N</b>) virus, or were mock infected (<b>Mock</b>). Supernatants from the culture were collected at various time points post infection. (<b>A</b>) Supernatants collected at 1, 12, 24, 48, and 72 hpi were determined for viral titers by plaque assay. Representative growth kinetics of the <b>WT</b> and <b>D701N</b> viruses in one donor cells are shown. Supernatants collected at 24 hpi were analyzed by ELISA for the levels of (<b>B</b>) IFN-λ, (<b>C</b>) CCL5, (<b>D</b>) IL-6, and (<b>E</b>) IL-8. Each symbol represents an average value of 2 or 3 repeats using the same cells from one donor and thus seven different symbols represent the data from seven independent experiments performed using cells from seven donors. Due to the variation in cytokine responses among different donors, for each donor, cytokine/chemokine induced by <b>WT</b> was set as 100% and cytokine/chemokine induced by <b>D701N</b> or <b>Mock</b> was presented as a percentage of <b>WT</b>. Horizontal bar in each group indicates the average value from seven donors and * indicates a statistical difference between WT and D701N (p<0.05, ANOVA).</p

DAS181 inhibits pandemic IFV A(H1N1) viral replication in mice.

<p>BALB/c mice were inoculated with the pandemic influenza A(H1N1) virus A/Mexico/4108/2009 (1000 MID₅₀/mouse, <i>i.n.</i>). The mice were treated with PBS or DAS181 (0.3, 0.6, or 1 mg/kg) <i>q.d.x5</i>, beginning 6 hours post-infection. Lungs were harvested on day 3 and day 6 p.i.. Viral titer in lung homogenate was determined by standard plaque assay on MDCK cells. Values represent mean±SEM viral titer amongst 3 mice per group/day. PFU = plaque forming units. No virus was detectable on day 6 in the DAS181 1.0 mg/kg treatment group. Statistical significance from PBS treatment determined by ANOVA with Bonferroni post-test, * = p<0.05, ** = p<0.01, *** = p<0.001. The limit of detection is 100 PFU/ml.</p

DAS181 inhibits pandemic IFV A(H1N1): viral yield in 2009 A(H1N1) infected HAE culture.

<p>Well differentiated primary human airway epithelium cultures (HAE) were pretreated with DAS181 for 2 hours (at 10 µM) before infection with either A/Mexico/4604/2009 or A/California/04/2009 at indicated infection levels. After 24 hrs the apical wash was collected and viral yield was determined on MDCK cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007788#s4" target="_blank">Materials and Methods</a>. Values represent mean±SD of quadruplicate analysis of each sample.</p

DAS181 inhibits pandemic IFV A(H1N1): Immunofluorescent detection of 2009 A(H1N1) foci in MDCK cells.

<p>MDCK cells were pretreated with DAS181 for 2 hours (at indicated concentrations) before infection with either pandemic influenza A(H1N1) A/Mexico/4604/09 (Mexico) or A/California/04/09 (California). After 24 hrs post-infection, the cultures were fixed and processed for immunostaining to detect virus antigen as marker of infected cells. White spots indicate infected cells that form foci (clusters) of different size ranging from 5 to 100's of individual infected cells.</p

DAS181 inhibits pandemic IFV A(H1N1) viral replication in <i>ex vivo</i> human bronchi tissue.

<p>Human bronchi tissues were collected from patients undergoing lung resection and immediately placed on air-liquid interface membranes for culturing. Within 12 hours the tissues were pretreated with PBS (“control”) or DAS181 (10 µg/cm², “2 h DAS181 pretreatment”) for 2 hours at 37°C. After washing, the tissues were infected with pandemic influenza A(H1N1) A/California/04/2009 (10⁶ TCID₅₀/tissue) for 1 hr and then washed. Tissues were then incubated at 37°C for 24 or 48 hrs before washing the apical surface and determining viral titer in either the apical wash by TCID₅₀ on MDCK cells (top panel), or within tissue homogenate by RT-PCR analysis of viral M-gene RNA level normalized to cellular β-actin RNA level (bottom panel). Dotted line represents limit of detection of the TCID₅₀ assay.</p

DAS181 inhibits pandemic IFV A(H1N1)-induced body weight loss in mice.

<p>BALB/c mice were inoculated with the pandemic influenza A(H1N1) virus A/Mexico/4108/2009 (1000 MID₅₀/mouse, <i>i.n.</i>). The mice were treated with PBS or DAS181 (0.3, 0.6, or 1 mg/kg) <i>q.d.x5</i>, beginning 6 hours post-infection. Body weight was measured daily for 14 days p.i., and is expressed normalized to starting body weight. The data is collected from the same animals/study shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007788#pone-0007788-g004" target="_blank">Figure 4</a>. Values represent mean±SEM body weight change amongst 10 mice per group. Statistical significance from PBS determined by analysis of Area Under Curve (AUC) with ANOVA and Bonferroni post-test, * = p<0.05, *** = P<0.001.</p