Reassortment findings after eight serial passages.

<p>Co-infection was performed in (A) PK-1 cells and (B) NSBE cells. While pdm09 and swH3N2 parent viruses were evident the only reassortant constellation identified was the PA segment of human pdm09 (light colored, “Hu”) within seven gene segments from swH3N2 (dark colored, “Sw”). Mixed gene segments with Ct values <8 are depicted as hash marks. Serial passaging was done in triplicate with a starting MOI of either 1.0 or 0.1 for each virus as indicated. To the left of (A) PK-1 cells and (B) NSBE cells is the kinetics of PA acquisition and loss for a MOI = 1∶1. Depicted is the first replicate shown from matrix. (C) 12 plaques with PA gene of pdm09 and 5 segments of swH3N2 from reassortant studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone-0110264-g001" target="_blank">Fig. 1</a>) were subjected to qPCR do delineate the final two segments, NP and PB1. Depicted on right, 11 of 12 reassortants were determined to be swH3N2-huPA, and 1 of 12 reassortants to be swH3N2-huPA-PB1.</p

qPCR analysis of the type I and III IFN response in dNSBE and dNHBE cells.

<p>The qPCR fold-changes at 8 h, 18 h, 24 h, 48 h, and 72 h pi are shown for virus-infected dNSBE (left) and NHBE cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone.0110264-Shu1" target="_blank">[4]</a>. Shown is type I interferons (A) IFN-α (B) IFN-β and type III interferons (C) IL-29 (D) IL-28. Time-points that were significant (*  =  p<0.05) by one-way ANOVA comparing swine and human gene expression are indicated. Fold-changes and standard deviations were calculated from Ct values run in quadruplicate relative mock and the housekeeping gene HPRT1.</p

Peak virus titers from plaque isolates with pdm09, swH3N2, swH3N2-huPA, and swH3N2-huPA-PB1 gene constellations.

<p>The cell lines used are: (A) PK-1 cells, (B) NSBE cells, and (C) MDCK cells. Significant (p<0.05) differences in viral titer for swH3N2-huPA and both parental strains is indicated by an asterisk (*). Of note, only one swH3N2 reassortant with huPA-PB1 genes was identified (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone-0110264-g002" target="_blank">Fig. 2C</a>). All infections used a MOI = 0.01. (D) Plaque diameter were measured 96 h pi in 2% agarose overlay solution. Cumulative plaque total measured from 2–3 separate six-well plates with 10–20 total plaques dilutions per well. * denotes significant difference between pdm09 and swH3N2-huPA.</p

Reassortment outcomes following human H1N1 pdm09 and swine H3N2 co-infection.

<p>Segments originating from pdm09 origin (light colored) and swH3N2 (dark-colored) are shown after co-infection in NSBE cells (left) and PK-1 cells. Primer and probes specific to parental strains pdm09 and swH3N2 gene segments were used and from left to right in the matrix correspond to PA, PB2, M, NS, NA and HA. All reassortant plaques identified are categorized by HA and NA backbone type. In total, 121 reassortants for PK-1 cells and 45 reassortants for NSBE cells were evaluated. The number of virus plaques showing 6-gene non-reassortment and the top 6 reassortant types are summarized below profile matrix.</p

qPCR analysis of IFN-stimulated genes (ISGs).

<p>Expression of ISG15 (left), MX1 (middle) and OAS1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110264#pone.0110264-Shu1" target="_blank">[4]</a> was determined at (A) 24 h and (B) 72 h pi where “S” denotes swine dNSBE and “H” denotes human dNHBE cells. Fold-changes and standard deviations were calculated from Ct values run in quadruplicate relative mock and the housekeeping gene HPRT1. Time points that were significant between NSBE and NHBE cells are noted (*  =  p<0.05).</p

Antiviral Efficacy of Verdinexor <i>In Vivo</i> in Two Animal Models of Influenza A Virus Infection

<div><p>Influenza A virus (IAV) causes seasonal epidemics of respiratory illness that can cause mild to severe illness and potentially death. Antiviral drugs are an important countermeasure against IAV; however, drug resistance has developed, thus new therapeutic approaches are being sought. Previously, we demonstrated the antiviral activity of a novel nuclear export inhibitor drug, verdinexor, to reduce influenza replication <i>in vitro</i> and pulmonary virus burden in mice. In this study, <i>in vivo</i> efficacy of verdinexor was further evaluated in two animal models or influenza virus infection, mice and ferrets. In mice, verdinexor was efficacious to limit virus shedding, reduce pulmonary pro-inflammatory cytokine expression, and moderate leukocyte infiltration into the bronchoalveolar space. Similarly, verdinexor-treated ferrets had reduced lung pathology, virus burden, and inflammatory cytokine expression in the nasal wash exudate. These findings support the anti-viral efficacy of verdinexor, and warrant its development as a novel antiviral therapeutic for influenza infection.</p></div

Verdinexor administration is efficacious when administered post-infection in mice.

<p>a) Mice were i.n. infected with 10×MID₅₀ of mouse-adapted A/California/04/09 and orally treated with 20 mg/kg verdinexor at day 1 and 3, day 2 and 4, day 3 and 5, day 4 and 5, or 10 mg/kg oseltamivir twice daily at day 1–4 post-infection. Lungs were harvested at day 6 post-infection for virus titration. b) Mice were i.n. infected with 10×LD₅₀ mouse-adapted A/California/04/09 and orally treated with 5 or 10 mg/kg verdinexor at day 1 and 3 or 10 mg/kg oseltamivir twice daily for 4 days starting at day 1 post-infection. Mice were evaluated for morbidity and mortality. Day of death was recorded either when a mouse was found deceased or when weight and health score required euthanasia. The data are from 8–10 mice per experimental group. c) Mice were infected with 10×MID₅₀ of A/Philippines/2/82/X-79 (H3N2). Mice were treated orally with 20 mg/kg verdinexor (V20) on both day 2 and 4 following infection, alone or in combination with 1 (O1) or 10 mg/kg (O10) oseltamivir twice daily on days 1 to 4. At day 5, mice were euthanized and lungs were collected for virus titration. The data are from 8–12 mice per experimental group. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001; ****, <i>p</i>≤0.0001.</p

Pharmacokinetic parameters of verdinexor following a <i>per os</i> dose of 5 mg/kg in male ferrets.

<p>Pharmacokinetic parameters of verdinexor following a <i>per os</i> dose of 5 mg/kg in male ferrets.</p

Verdinexor reduces inflammatory cell infiltration into the bronchoalveolar space in influenza infected mice.

<p>Mice were infected with 10×MID₅₀ of mouse-adapted A/California/04/09 intranasally and treated orally with 20 mg/kg verdinexor at day 1 and 3 or 10 mg/kg oseltamivir twice daily post-infection. BAL cells from day 2 and 4 post-infection were characterized using flow cytometry. Percent of CD11c+Gr-1-, CD11c+Gr-1+, CD3+, DX5+, and CD3+DX5+ cells were plotted. The data are from five mice per experimental group per time point. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001; ****, <i>p</i>≤0.0001.</p

Verdinexor reduces influenza virus shedding and pro-inflammatory cytokines expression in BAL fluid.

<p>a) Experimental design: mice were infected with 10×MID₅₀ of mouse-adapted A/California/04/09 intranasally and treated orally with 20 mg/kg verdinexor at day 1 and 3 or 10 mg/kg oseltamivir twice daily post-infection. Five mice per group were sacrificed at day 2 and 4 post-infection and BAL fluids were collected. b) Virus titers in the BAL were evaluated. Protein levels of c) IFN-γ, d) TNF-α, e) IL-6, f) IL-12p40, g) MCP-1, and h) RANTES in the BAL fluid were evaluated using the mouse cytokines/chemokines multiplex assay kit. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01; n.s., not significant.</p