Detailed histopathological scores of all animals examined in this study.

Individual clinical scores for alveolitis, bronchiolitis, perivascular cuffing (PVC), vasculitis, interstitial pneumonia (IP), and pleuritis. Columns show data medians, symbols represent individual animals; 1-way ANOVA with Tukey’s post hoc test; p values are specified; n values are specified in Fig 4F. (TIF)</p

All statistical analyses performed for this study.

All statistical analyses performed for this study.</p

Urea-PAGE fractionation (uncropped) of RNA transcripts.

a) Template and primer used in the reaction. b-e) Independent repeats of primer extension assay by IAV polymerase. Dashed rectangle shows insert presented in Fig 1E. Replicates were included in quantitations presented in Fig 1E and 1F. (TIF)</p

Clinical signs of mice involved in mitigation of histopathology study.

a-b) Body weight measurements taken twice daily (a), and rectal body temperature determined once daily (b). Lines intersect, and symbols show, data means ± SD. Dashed line in (a) specifies predefined endpoint. (TIF)</p

Efficacy of 4’-FlU in an immunocompromised host and against HPAI.

a) Efficacy study schematic in immunocompromised mice, lacking B and T cells (RAG1 KO) or IFN1 receptor function (IFNar1 KO). b) Survival study of animals treated as shown in (a). Median survival time in days is specified; Kaplan-Meier simple survival analysis. c) Lung viral load in animals from (a), determined 4.5 days after infection. d) Dose-response minigenome assay with RdRP complexes derived from IAV subtypes H1N1, H5N1, and H7N9. Lines represent 4-parameter variable slope regression models; symbols show data medians with 95% CI; n = 3. EC50 concentrations and 95% CI are specified. e) Efficacy study schematic of 4’-FlU against HPAI H5N1. f) Body weight measurements of animals shown in (e). Lines intersect, and symbols show, data means ± SD, normalized to animal body weight at study start. Dashed line shows predefined endpoint. g) Survival study of animals infected with HPAI and treated as shown in (e). Median survival time in days is specified; Kaplan-Meier simple survival analysis. h) Lung virus load on day 3 after infection. Columns in (c,h) represent geometric means ± SD; symbols show individual animals; 1-way ANOVA with Dunnett’s post hoc test; P values and n values are specified.</p

Clinical signs in 4’-FlU-treated mice used in immune response-mitigation study.

a-b) Body weight measurements taken twice daily (a), and rectal body temperature determined once daily (b). Lines intersect, and symbols show, data means ± SD; n values are specified in Fig 4. Dashed line in (a) specifies predefined endpoint. (TIF)</p

Effect of 4’-FlU on the antiviral immune response and lung histopathology.

a)In vivo Bioplex and histopathology study schematic. b) Lung viral titers in animals treated as in (a). c-d) Changes in IL-6 (c) and TNF-α (d) levels present in BALF of animals treated as in (a), relative to levels at time of infection. Lines in (b) intersect, and symbols show, geometric means ± SD, lines in (c-d) intersect, and symbols show, data medians with 95% CI; 2-way ANOVA with Tukey’s post hoc test; P values are given. e) Representative photomicrographs of lung tissue extracted 5 days after infection of animals treated as in (a). Tissues of two individual animals per study arm are shown at 10× magnification; scale bar denotes 100 μm; Br, Bronchiole; Bl, Blood vessel. f) Histopathology scores of animals treated as in (a). Lungs were extracted 5 days after infection. Scores for each animal represents a mean of individual alveolitis, bronchiolitis, vasculitis, pleuritis, perivascular cuffing (PVC), and interstitial pneumonia (IP) scores. Columns represent data medians with 95% CI; symbols show mean scores for each individual animal; 1-way ANOVA with Dunnett’s post hoc test, P values and n values for each study arm are specified.</p

Clinical signs of mice infected and reinfected with pdmCa09.

a-d) Body weight measurements taken twice daily (a, c), and rectal body temperature determined once daily (b, d). Lines intersect, and symbols show, data means ± SD. Results are shown for animals after the original infection (a-b) and after homotypic reinfection (c-d). Dashed lines in (a,c) specify predefined endpoint. (TIF)</p

Treatment paradigms of 4’-FlU in mice.

a) Mouse plasma exposure after a single oral dose of 1.5 mg/kg bodyweight. The red area denotes cell culture EC90 ± 1 × SD against WSN, all donors as shown in (S1A Fig). b) Tissue distribution of bioactive anabolite 4’-FlU-TP in animals shown in (a) 12 hours after dosing. c) Schematic of dose-to-failure study against standard recA/CA/2009 (H1N1). d) Survival of animals treated as specified in (c). Median survival (med. survival) time in days is specified; Kaplan-Meier simple survival analysis. e) Study schematic to determine the therapeutic time window of oral 4’-FlU in mice, using recA/CA/2009-maxGFP-HA (H1N1) as viral target. f) Survival of animals treated as specified in (e). Median survival time in days is specified; Kaplan-Meier simple survival analysis. g) Lung viral load of a set of animals treated as in (e) was determined 4.5 days after infection. h) Time-to-viral-clearance study. Mice were infected and treated with 4’-FlU at 2 mg/kg starting 24 hours after infection and continued q.d. Lung virus load was determined in vehicle-treated animals 4.5 days after infection, and in 4’-FlU-treated animals 4.5, 7, and 9 days after infection. i) Schematic of minimal-number-of-doses finding study. j) Lung viral load of animals treated as in (i), determined 4.5 days after infection. k) Survival of a set of animals treated as in (i). Median survival time in days is specified; Kaplan-Meier simple survival analysis. Columns in (b) represent data medians with 95% CI, columns in (g-h,j) represent geometric means ± SD; symbols specify individual animals; statistical analysis in (g-h,j) with 1-way ANOVA with Dunnett’s post hoc test.</p

<i>In vitro</i> potency and MOA of 4’-FlU against influenza viruses.

a) Structure of 4’-FlU. b) Dose-response assay of 4’-FlU on MDCK cells. Cells were infected with different subtype IAVs or different IBV isolates. c) 4’-FlU antiviral activity in 2D HAE cultures. Dose-response assays of 4’-FlU against A/California/07/2009 (H1N1), A/swine/Spain/53207/2004 (H1N1), A/Wisconsin/67/2005 (H3N2), B/Memphis/20/1996 (Yamagata lineage), and B/Malaysia/2506/2004 (Victoria lineage) on undifferentiated HAEs derived from a healthy female donor. Lines in (b-c) intersect, and symbols show, geometric means ± SD (n = 3); EC50 and EC90 values based on 4-parameter variable slope regression modeling are given. d) Coomassie blue staining of purified recombinant IAV RdRP proteins after gel electrophoresis and RNA template sequences used in primer extension assays. e) In vitro RdRP assay in the presence of 32P-ATP, CTP, GTP, and UTP or 4’-FlU-TP as indicated. Representative autoradiogram showing the sequence section highlighted by dashed box in (d); the first incorporation of UTP or 4’-FlU-TP is after position i = 8 of the amplicon (arrow). The sequence of the amplicon and results of phosphoimager-quantitation of relative signal intensities observed in the presence of 11 μM UTP or 4’-FlU-TP are specified to the left and right of the autoradiogram, respectively. Quantitation graph shows mean values of independent experiments (n = 3) ± SD; analysis with 1-way ANOVA with Dunnett’s post hoc test; P values are shown in the graph. Uncropped autoradiogram and replicates are provided in (S2 Fig). F) Kinetic analysis of 4’-FlU-TP and UTP incorporation into the amplicon shown in (e) and (S2 Fig). Lines represent non-linear regression kinetics with Michaelis-Menten model, Km and Vmax are shown, error bars represent 95% CI.</p