Bootstrapped ML trees, inferred with RAxML, for regions of the PA segment of avian H5N1 sequences.

<p>Tree A was inferred for the region defined by positions 1–1361 & 1939–2314. Tree B was inferred for the region defined by positions 1362–1938. The red sequence is the putative recombinant sequence. Blue and green sequences are major and minor parental sequences, respectively, as identified by 3SEQ. Trees are midpoint rooted. ML trees inferred with PAUP* have some non-critical differences the two subclades marked with open circles.</p

False positives of 3SEQ algorithm on simulated clonal data sets of the same size and diversity as in this study.

<p><b>Footnote:</b> P-value in the column heading is the Dunn-Sidak corrected P-value.</p

Summary of 18 datasets used in this study.

<p><b>Footnotes:</b> P-values are corrected with a Dunn-Sidak correction for the numbers of triplets tested in each dataset. P-values are shown as simple orders of magnitude when P<10⁻⁶.</p

Bootstrapped ML trees for the PA segment of avian H5N1 sequences.

<p>Tree A was inferred for the region defined by positions 1–139 & 2016–2314. Tree B was inferred for the region defined by positions 140–2015. ML trees inferred with PAUP* have some non-critical differences the two subclades marked with open circles. Phylogenetic relationships in these trees do not support a hypothesis of homologous recombination. Other features as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010434#pone-0010434-g001" target="_blank">Figure 1</a>.</p

TCAD therapy is highly efficacious in mice.

<p>(A) The kinetics of A/H5N1 virus replication in mouse lungs. Mice were infected with an LD₁₀₀ dose of virus and the lungs were harvested at the indicated time points (N = 5), homogenized, and the virus titer was determined by endpoint titration in Madin-Darby canine kidney cells. Kaplan Meier survival curves for treatment of mice infected with lethal doses of (B) a drug susceptible A/H5N1 influenza virus (A/Duck/MN/1525/81) and (C) AMT-resistant 2009 A/H1N1 influenza virus (A/California/04/09). Maximum weight loss analysis for the treatment of mice infected with (D) drug susceptible A/H5N1 and (E) AMT-resistant 2009 A/H1N1. For this experiment, mice were treated with AMT (46 mg/kg/day), OSL (25 mg/kg/day), and RBV (27 mg/kg/day) as monotherapies and in double or triple combinations at the same doses. Treatments were given three times a day for 5 days starting 24 hours after virus challenge, and survival and body weight loss were monitored over 21 days.</p

Statistical comparison of Kaplan-Meier survival curves for treatment of mice infected with wild type A/H5N1 influenza.

<p>(-) No statistical comparison performed.</p

Statistical comparison of Kaplan-Meier survival curves for treatment of mice infected with AMT-resistant 2009 A/H1N1 influenza.

<p>(-) No statistical comparison performed.</p

The effects of delayed treatment in mice.

<p>Mice were infected with A/H5N1 and treated with (A) TCAD therapy or (B) OSL monotherapy. Treatments were given three times a day for 5 days starting at -4 (prophylaxis), 24, 48, or 72 hours after virus challenge, and survival and (C) maximum body weight loss were monitored over 21 days. Mice were treated with OSL (25 mg/kg/day) alone, or TCAD [AMT (46 mg/kg/day), OSL (25 mg/kg/day), and RBV (27 mg/kg/day)]. *P<0.05 versus placebo; *** P<0.05 versus OSL at the same treatment time point.</p

The activity of AMT is enhanced in combination with OSL and RBV against drug susceptible and resistant viruses.

<p>(A,B,C) Mice infected with AMT-susceptible A/H5N1 were treated with escalating doses of AMT (0, 15, and 46 mg/kg/day) either as a single agent or in the context of TCAD therapy (with 25 mg/kg/day OSL and 27 mg/kg/day RBV). (D,E,F) Mice infected with AMT-resistant 2009 A/H1N1 were treated with escalating doses of AMT (0, 46, and 138 mg/kg/day) either as a single agent or in the context of TCAD therapy. Kaplan-Meier survival curves for (A) A/H5N1 or (D) 2009 A/H1N1 infection. *P<0.05 versus no AMT, ***P<0.05 versus low dose AMT. Distribution of maximum body weight loss over the course of infection for (B) A/H5N1 influenza or (E) 2009 A/H1N1 influenza. Data bars represent median +/− interquartile range. The mean percentage weight change at day 5 in mice infected with lethal doses of (C) A/H5N1 influenza and (F) 2009 A/H1N1 influenza. Data bars represent mean +/− standard deviation.</p

Statistical comparison of percent maximum weight loss for treatment of mice infected with wild-type A/H5N1 influenza.

<p>(-) No statistical comparison performed.</p