4’-FlU is an immediate chain terminator of influenza virus polymerase

Through biochemical RdRP assays using purified recombinant RSV and SARS-CoV-2 polymerase complexes and synthetic template RNA, we have demonstrated that incorporation of 4’-FlU triphosphate (4’-FlU-TP) into nascent chain RNA triggers delayed termination of the RNA-dependent RNA polymerases from a negative sense (RSV) and a positive sense (SARS-CoV-2) RNA virus. Whereas variations in stalling efficiencies were observed between sequences and polymerase, position i+3 marked the predominant stalling site [24]. Following expression of IAV RdRP PA, PB1, and PB2 subunits in insect cells, we subjected affinity chromatography purified, reconstituted IAV polymerase to the equivalent biochemical RdRP assay in the presence of increasing concentrations of 4’-FlU-TP or UTP (Figs 1D, 1E and S2A to S2E). 4’-FlU-TP was incorporated instead of UTP when the polymerase reached the first adenosine in the template RNA as we had noted before in RSV and SARS-CoV-2 RdRP assays [24], but then triggered immediate chain termination of IAV RdRP. Densitometric quantitation of relative amplicon intensities to compare incorporation rates of the analog relative to UTP revealed an approximately 4-fold higher affinity of IAV polymerase for endogenous UTP than for 4’-FlU-TP (Fig 1F).

These results demonstrate high sensitivity of influenza virus polymerases to 4’-FlU and identify sequence-independent immediate chain termination at position i as a predominant mechanism of influenza virus RdRP inhibition by the compound.

Pharmacokinetic (PK) properties of 4’-FlU in mouse plasma and tissues

We have previously demonstrated oral bioavailability of 4’-FlU in ferrets and efficient intracellular anabolism to 4’-FlU-TP with prolonged tissue exposure levels [24]. To assess cross-species conservation of PK profiles between IAV efficacy models used in this study, ferrets and mice, we subjected the compound to a single-dose oral PK study in mice, measuring 4’-FlU plasma concentrations and corresponding 5’-triphosphate levels in selected soft tissues after oral dosing at 1.5 mg/kg body weight (Fig 2A and 2B). Free 4’-FlU reached a maximal plasma exposure of approximately 1 μM 90 minutes after oral administration, far exceeding the cell culture EC₉₀ concentration against the influenza virus test panel. Plasma levels were sustained high over a 12-hour period after dosing. Corresponding 4’-FlU-TP tissue exposure levels 12 hours after dosing were approximately 1 nmol/g tissue in all organs but brain and kidney, underscoring suitability of 4’-FlU for in vivo efficacy testing against IAV.

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Fig 2. 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.

https://doi.org/10.1371/journal.ppat.1011342.g002

Late-onset treatment with 4’-FlU mediates complete survival of IAV-infected animals

For mouse efficacy studies, animals were infected intranasally with a lethal inoculum amount of pdmCa09, which replicates efficiently in mice without a requirement for species adaptation [28]. Infection results in severe viral pneumonia with major clinical signs such as weight loss and hypothermia. To establish the lowest efficacious dose, we examined 3 4’-FlU dose levels in a survival study, each administered by oral gavage on three consecutive days in a once daily (q.d.) regimen starting at the time of infection (Fig 2C). All animals treated at the 2 mg/kg dose level survived, whereas all mice of the vehicle-treated and lowest dose (0.08 mg/kg) groups succumbed to infection within 5 days of inoculation (Fig 2D) and experienced clinical signs (S3A and S3B Fig). Animals receiving the intermediate 4’-FlU dose (0.4 mg/kg) had prolonged survival, but ultimately all died.

Having established 2 mg/kg 4’-FlU administered q.d. as a low efficacious dose, we next determined the latest time for initiation of effective treatment at this dose level (Fig 2E). All vehicle-treated animals again succumbed to infection within 5 days of inoculation (Fig 2F). In contrast, treatment start at 2.5 days after infection, which corresponds to 0.5 days after the onset of major clinical signs (S3C and S3D Fig) ensured complete survival, and 80% of animals of a group receiving the first dose 3 days after infection survived, outlining a broad therapeutic time window for treatment of influenza with 4’-FlU. Therapeutic benefit of late-onset treatment was corroborated by a significant reduction in lung virus load compared to vehicle-treated controls at 4.5 days after infection, determined in a parallel set of equally treated animals (Fig 2G). Within 7 days, virus became undetectable in lungs of animals that were treated starting 24 hours after infection with oral 4’-FlU (2 mg/kg q.d.; 3 doses total), indicating that viral replication had ceased (Fig 2H). No rebound of virus replication was observed within a 9-day period after infection (S3E and S3F Fig). These results define a broad therapeutic time window for effective treatment of IAV infection with 4’-FlU.

To determine the minimal number of oral 4’-FlU doses (2 mg/kg q.d.) for complete survival, we again initiated treatment 24 hours after infection, in this study comparing the effect of a single vs double or triple doses (Fig 2I). Lung virus load was assessed 4.5 days after infection and survival monitored in a parallel set of equally treated animals. The number of doses administered correlated with antiviral effect size, but even a single dose of 4’-FlU resulted in a statistically significant reduction in lung virus load of approximately one order of magnitude (Fig 2J). Reduced viral burden translated to survival of some animals in the single and double dose groups. However, three doses were required for full therapeutic benefit (Fig 2K) and only mild clinical signs (S3G and S3H Fig).

4’-FlU effect on IAV transmission in the ferret model

Since mice, in contrast to ferrets, do not shed IAVs, we next explored anti-IAV efficacy of 4’-FlU in the ferret model. Guided by the dosing regimen established in mice, we treated animals infected intranasally with pdmCa09 with either 2 mg/kg (standard dose) or 0.5 mg/kg (low dose), administered orally starting 24 hours after infection (Fig 3A). Animals received one (2 mg/kg level) or two doses (either 2 or 0.5 mg/kg level) total. Fever is the predominant clinical sign associated with pdmCa09 IAV infection in ferrets. Monitoring body temperature continuously telemetrically, we observed significant reduction in all treated animals within 24 hours of the first dose, whereas vehicle-treated animals showed a prolonged fever plateau (S4A Fig). Viral titers in nasal lavages were statistically significantly reduced compared to those of vehicle animals, but effect size was lower in animals of the low dose than the standard dose group, and virus rebounded 2.5 days after infection in the single standard dose group (Fig 3B). In contrast, two standard doses 4’-FlU given q.d. were sterilizing within 2 days of treatment initiation. Virus load in nasal turbinates assessed 4 days after infection corroborated the lavage titers, revealing a dose-dependent significant antiviral effect of two doses, whereas a single dose had no significant effect on virus titer in turbinates at the time of tissue harvest (Fig 3C). Examination of lower respiratory tract tissues, trachea, and bronchoalveolar lavage fluid (BALF) revealed, however, that a single standard dose of 4’-FlU administered at the time of onset of clinical signs completely suppressed the development of viral pneumonia (Fig 3D).

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Fig 3. In vivo efficacy of 4’-FlU in the ferret transmission model.

a) Ferret efficacy study schematic. b) Ferret shed viral titers in nasal lavages (n = 3). c-d) Tissue and BALF viral titers determined 4 days after infection. e) Transmission study schematic. Treatment of source ferrets was started 12 or 24 hours after infection, animals were co-housed with untreated contact ferrets 2.5 days after infection. f) Ferret shed viral titers in nasal lavages of source animals and their sentinels. Light blue dashed line marks time of treatment onset; orange box highlights co-housing period. g-h) Virus load in nasal turbinates (g) and lung and trachea tissues (h) in source animals, collected 4 days after infection, respectively. Lines in (b,f) intersect, and symbols show, geometric means ± SD; 2-way ANOVA with Tukey’s post hoc test, P values are specified (in (f), P values in black compare source animals, P values in blue and brown compare contact ferrets). Columns in (c-d,g-h) represent geometric means ± SD; symbols show individual animals; 1-way ANOVA with Dunnett’s post hoc test (c-d) or unpaired t-test (g-h); P values are specified.

https://doi.org/10.1371/journal.ppat.1011342.g003

To determine the effect of 4’-FlU on virus spread, we treated in a direct-contact transmission study source ferrets infected with pdmCa09 with the compound as before, starting 12 or 24 hours after infection (Fig 3E). Co-housing of source ferrets with untreated sentinels in a 1:1 ratio was initiated 2.5 days after study start. pdmCa09 rapidly spread from vehicle-treated source ferrets to their direct contacts, which first became virus-positive in nasal lavages only 12 hours after the beginning of co-housing (Fig 3F) and had progressed to viral pneumonia with high turbinate, lung and trachea virus load by study end (Fig 3G and 3H) and clinical signs (S4B to S4E Fig). Treatment of source animals with 4’-FlU initiated 12 or 24 hours after infection in each case resulted in exponential decline in shed virus load, but virus was undetectable in lavages of the 12-hour treatment group by the time of co-housing whereas approximately 10⁴ TCID₅₀ units/ml lavage remained detectable in lavages of the 24-hour treatment group at that time. We did not detect any virus transmission to untreated sentinels of the 12-hour group. However, low-grade transmission to direct contacts of the 24-hour treatment group occurred, characterized by delayed onset of virus shedding compared to sentinels of vehicle-treated source animals and a reduced peak shed virus load (Fig 3H). Shedding from all sentinels that became virus-positive ceased approximately 12 days after the beginning of co-housing. These results confirm cross-species anti-IAV efficacy of 4’-FlU, reveal that a single oral dose is sufficient to prevent advance to severe influenza, and demonstrate a shortened window in which a treated case remains fully infectious.

Mitigation of IAV pathogenesis and lung histopathology by 4’-FlU

To better understand the impact of 4’-FlU on mitigating immunopathogenesis, the major driver of clinical signs associated with influenza virus infection, we determined inflammatory cytokine profiles for a 10-day period after infection of mice with pdmCa09 using a 6-plex Th17 cytokine assay (Fig 4A). Treated animals received the 3-dose standard 4’-FlU regimen as before, started 24 hours after infection. Lung tissue for histopathology was extracted from parallel sets of equally treated animals 5 or 21 days after infection. Lung titers in vehicle-treated animals demonstrated fast-onset viral pneumonia, approaching a burden of 10⁶ infectious units/g lung tissue 4 days after infection, when all animals of the vehicle group had succumbed to infection with major clinical signs (Figs 4B, S5A and S5B). Pro-inflammatory cytokines rapidly increased in the BALF of these animals, revealing a very strong antiviral response until death of the vehicle animals as expected after infection with pdmCa09 (Figs 4C, 4D and S6). Treatment with 4’-FlU again efficiently suppressed virus replication within 24 hours of the first dose (Fig 4B). Coinciding with the reduction in virus burden of treated animals by approximately two orders of magnitude by day 3 after infection, the proinflammatory response was greatly alleviated (Figs 4C, 4D and S6) and all treated animals survived (S5A Fig).

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Fig 4. 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.

https://doi.org/10.1371/journal.ppat.1011342.g004

Consistent with a better clinical outcome (S7A and S7B Fig), lowered viral load, and mitigated innate antiviral response in 4’-FlU treated vehicle animals, analysis of lung histopathology 5 days after infection revealed reduced lung lesions and alleviated cellular infiltrates in lung tissue of treated animals (Figs 4E and S8). Overall combined histopathology scores of individual animals representing the degree of cellular infiltrates into alveoli and bronchioles, the extent of perivascular cuffing, and the severity of interstitial pneumonia, pleuritis, and vasculitis were statistically significantly lower in the treatment than vehicle group (Figs 4F and S9).

4’-FlU control of influenza in immunocompromised hosts and of HPAI infection

In the clinic, the risk of progression to severe disease and poor overall outcome is greatly increased when immunocompetence of the patient is impaired or after zoonotic spillover of HPAIs into the human population [29,30]. To first assess the potential of 4’-FlU to pharmacologically manage influenza in high-risk hosts, we infected interferon α/β receptor knockout (IFNAR1 KO) and V(D)J recombination activation gene RAG-1 KO mice with pdmCa09. Whereas the former cannot mount an effective type I IFN innate response [31,32], the latter are compromised in their cell-mediated adaptive immunity due to a lack of mature T and B cells [33]. Oral treatment with 4’-FlU was initiated 24 hours after infection at standard and a high-dose (10 mg/kg) level, then continued q.d. for a total of 7 doses of each group (Fig 5A). Independent of 4’-FlU dose level, all treated animals of the RAG-1 KO and IFNAR1 KO groups survived, whereas all animals in the vehicle-treated groups developed severe clinical signs (S10A to S10D Fig) and succumbed to infection within 4 days (Fig 5B). Reduction in lung virus load of treated vs vehicle animals after three q.d. 4’-FlU doses (day 4 after infection) was dose level-dependent, but even at the standard dose, this reduction exceeded two orders of magnitude in either knockout mouse strain (Fig 5C).

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Fig 5. 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.

https://doi.org/10.1371/journal.ppat.1011342.g005

Next, we explored 4’-FlU efficacy against HPAIs, initially testing potency against polymerases derived from A/CA/07/2009 (H1N1), A/VN/12/2003 (H5N1), and A/Anhui/1/2013 (H7N9) in minireplicon dose-response assays (Fig 5D). The compound dose-dependently inhibited activity of either HPAI-derived polymerase complex, reaching active concentrations resembling that observed against pdmCa09 RdRP. Selecting A/VN/12/2003 (H5N1), which causes lethal encephalitis in mice [34], for a proof-of-concept efficacy study of 4’-FlU against zoonotic HPAIs, we started treatment at the 5 mg/kg dose level 12 or 24 hours after infection and continued q.d. thereafter (Fig 5E). This elevated dose was chosen to compensate for lower brain tissue exposure of 4’-FlU (Fig 2A) when treating infection by a CNS-invasive IAV. None of the animals of either 4’-FlU treatment group developed clinical signs even when treatment was initiated 24 hours after infection (Fig 5F), and all treated animals survived (Fig 5G). In contrast, 19 out of 20 vehicle-treated animals succumbed to A/VN/12/2003 (H5N1) with a median survival time of 9 days after infection, developing clinical signs on day 7 after infection followed by rapid further deterioration (Fig 5F). Vehicle-treated animals experienced high lung virus loads three days after infection (Fig 5H). Treatment reduced HPAI burden by several orders of magnitude to limit of detection. These results confirm uncompromised efficacy of 4’-FlU in high-risk immunocompromised hosts and against highly pathogenic zoonotic viruses with high pandemic concern.

Anti-IAV immune status of 4’-FlU-treated recoveries

Highly efficacious antivirals may suppress host immune activation, especially when treatment is started early in the course of infection, which may leave the host susceptible to subsequent re-infection and may potentially even result in exacerbated disease [35–37]. To explore the effect of 4’-FlU on subsequent homotypic IAV rechallenge of recoveries, we infected animals with pdmCa09 and started standard 3-dose 4’-FlU treatment at three time points, therapeutically at 24 and 48 hours after infection and prophylactically at 12 hours before infection (Fig 6A). For this study, mice were inoculated with aerosolized virus in a temperature and airflow-controlled aerosol chamber using an Aeroneb nebulizer (S11A and S11B Fig) to better mimic natural infection. Prior to study start, we performed pilot experiments to determine suitable exposure time (S12A to S12C Fig), infectious dose (S13A and S13B Fig) and proof-of-concept for 4’-FlU efficacy (S14A to S14D Fig) after infection with aerosolized virus. Mirroring the outcome of our previous efficacy studies, animals of all treatment groups survived the original infection, whereas all animals of the vehicle group reached predefined clinical endpoints 9 days after infection and had to be terminated (Fig 6B) with major clinical signs (S15A and S15B Fig). To assess the status of humoral anti-Ca09 immunity, we collected blood samples from the treated recoverees 4, 8, and 12 weeks after the original infection and determined neutralizing antibody (nAb) titers. Mice in both therapeutic treatment groups mounted a robust humoral anti-IAV response, whereas no nAbs were detectable in the prophylactically treated animals 8 weeks after the original infection (Fig 6C). Upon homotypic rechallenge with aerosolized pdmCa09 10 weeks after the original infection, however, all 4’-FlU-experienced recoverees had a significant benefit over a new set of IAV-naïve mice (Fig 6D). All animals of the therapeutic groups survived with significant boost in nAb titers relative to week 8 levels. Remarkably, four of the five animals treated prophylactically with 4’-FlU at the time of the original infection also survived the homotypic rechallenge and mounted a robust humoral anti-Ca09 response by week 12. In contrast, all newly added IAV-inexperienced animals succumbed to infection within 9 days, confirming efficient virus delivery through aerosolization at rechallenge. Animals of the treatment group remained disease-free during rechallenge, and clinical signs were alleviated in mice of the prophylactic group (S15C and S15D Fig). Independent of the original 4’-FlU regimen applied, we did not detect any signs of exacerbated disease upon rechallenge of treatment-experienced recoverees.

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Fig 6. Reinfection of 4’-FlU-experienced animals with homotypic H1N1.

a) Schematic of the treatment and reinfection study. For infection and reinfection, mice received aerosolized pdmCa09. b) Survival study of animals infected, treated, and reinfected as shown in (a). Median survival time in days is specified; Kaplan-Meier simple survival analysis. c) Anti-H1N1 neutralizing antibody (nAb) titers developing in animals from (a). Lines intersect, and symbols show, geometric means ± SD; 2-way ANOVA with Tukey’s post hoc test; P values are specified. d) Survival of animals after re-infection. Median survival time in days is specified; Kaplan-Meier simple survival analysis. N numbers for animals in (a-d) are specified in (a).

https://doi.org/10.1371/journal.ppat.1011342.g006

Ethics statement

All animal work was performed in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act Code of Federal Regulations. Experiments involving mice and ferrets were approved by the Georgia State University Institutional Animal Care and Use Committee (IACUC) under protocols A20012 and A21020, respectively. In vivo experimentation with HPAI viruses was approved by the University of Georgia at Athens IACUC under protocol A2020 03–033. All experiments using infectious material were approved by the Georgia State University and the University of Georgia at Athens Institutional Biosafety Committees (IBCs) and performed in BSL-2/ABSL-2 or BSL-3/ABSL-3 containment facilities, respectively. Experiments involving highly pathogenic avian influenza (HPAI) viruses were reviewed and approved by the IBC of the University of Georgia at Athens and were conducted in enhanced biosafety level 3 (BSL3+) containment according to guidelines for the use of select agents approved by the Centers for Disease Control and Prevention (CDC).

Study design

Cells, ferrets and mice were used as in vitro, ex vivo and in vivo models, respectively, to examine efficacy of 4’-FlU against influenza infections. Biochemical RdRP assays were added for mechanistic characterization against the influenza virus target. Viruses were administered through intranasal or aerosolized (mice only) inoculation and virus load monitored periodically in nasal lavages (ferrets only), and in respiratory tissues of ferrets and mice extracted 4 days and 3–14 days, respectively, after infection. Virus titers were determined through TCID₅₀-titration.

Cells, viruses, and compound synthesis

MDCK (ATCC CCL-34) and 293T (ATCC CRL-3216) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% heat-inactivated fetal bovine serum (FBS) at 37°C and 5% CO₂. Human primary cells were grown in epithelial differentiation medium [24]. A/swine/Spain/53207/2004 (H1N1), A/Wisconsin/67/2005 (H3N2), A/California/07/2009 (H1N1)pdm09, A/Netherlands/602/2009 (H1N1)pdm09, A/WSN/33 (H1N1), A/Wyoming/03/2003 (H3N2), B/Memphis/20/1996 (Yamagata), and B/Malaysia/2506/2004 (Victoria) were propagated on MDCK cells using serum-free DMEM supplemented with 0.5% Trypsin. All influenza A viruses were grown at 37°C, influenza B viruses at 32°C. A/Viet Nam/1203/2004 (H5N1) and A/duck/Alberta/35/76 (H1N1) were propagated in the allantoic cavity of embryonated hen eggs at 37°C for 24 or 48 hours, respectively, clarified, aliquoted, and stored at −80°C. Virus stock titers were determined through TCID₅₀-titration or plaque assay (A/Viet Nam/1203/2004 (H5N1)), stocks were stored in aliquots at -80°C. Recombinant influenza viruses were recovered using an 8-plasmid system generated for A/CA/07/2009 (H1N1) that was based on original reports for laboratory-adapted IAV [59]. Reporter viruses were generated through insertion of a nanoLuc or maxGFP-encoding ORF in frame at the 5’-end of the viral HA ORF, separated by a 2A cleavage site as described for other recombinant IAVs [60]. Genetic stability of the resulting nanoLuc-HA and maxGFP-HA segments was validated through sequence confirmation after 10 consecutive viral passages on MDCK cells. 4’-FlU was synthesized from 5’-(3-chlorobenzoyloxy)-2’,3’-di-O-acetyl-4’-fluorouridine and 4N ammonia in methanol as previously described [24], authenticated through elemental analysis, and stored as dry powder. Working aliquots were dissolved in DMSO or 10 mM sodium citrate with 0.5% Tween 80 for in vitro and in vivo studies, respectively.

Virus titration

Virus samples were serially diluted (10-fold starting at 1:10 initial dilution) in serum-free DMEM supplemented with 0.5% Trypsin (Gibco). Serial dilutions were added to MDCK cells seeded in 96-well plate at 1×10⁴ cells per well 24 hours before infection. Infected plates were incubated for 72 hours at 37°C with 5% CO₂, followed by transfer of culture supernatants to suspension of chicken red blood cells and scoring of wells based on hemagglutination activity. A/Viet Nam/1203/2004 (H5N1) virus and samples were titered by plaque assay; serially diluted 10-fold in DMEM with 2% FBS, added to MDCK cells in 12-well plates and overlaid with DMEM+2% FBS with 1.2% Avicel microcrystalline cellulose. Plates were incubated for 48 hours at 37°C with 5% CO₂, then washed, fixed with methanol:acetone (80:20), and counter-stained with crystal violet to visualize plaques.

Dose-response assays

Active concentrations (EC₅₀ and EC₉₀) of 4’-FlU were calculated through 4-parameter variable slope regression modeling of dose-response assay data, derived from progeny virus yield (TCID₅₀) assays, luciferase reporter virus assays, or minigenome assays as specified. To test compound potency, 4-FlU was in all dose-response assays assessed in 3-fold serial dilutions, each tested in 3 independent repeats (biological repeats), with starting concentrations of 20 μM 4’-FlU. Control wells received vehicle (DMSO) volume equivalents. Dose-response assays on MDCK cells and 2D HAE cultures were carried out in 96-well plate format (1.5 × 10⁴ cells/well) or 24-well plate format (1.5 x 10⁵ cells/well), respectively. MDCK cells were infected in serum-free DMEM supplemented with 0.5% TPCK-treated trypsin (Gibco) with an MOI of 0.02 TCID₅₀ units/cell, HAE cultures were infected in BronchiaLife media (LifeLine) supplemented with 0.75 μg/ml TPCK-treated trypsin with an MOI of 1.0 TCID₅₀ units/cell. Culture supernatants were removed 24 (HAE cultures) or 36–48 hours (MDCK cells) after infection and viral titers determined through TCID₅₀ titration. For luciferase reporter viruses, relative luciferase activity was read in situ using a BioTek Synergy H1 multimode reader and signals normalized according to the formula % relative activity = (RLU_i−RLU_min)/(RLU_max−RLU_min) × 100, with RLU_i specifying sample luciferase value, RLU_min specifying values from equally transfected, vehicle-treated cells that did not receive the PB2 subunit encoding plasmid, and RLU_max specifying values from fully transfected, vehicle-treated cells.

Primary human airway epithelium cultures grown at air-liquid interface

Primary HAEs (3 × 10⁵/well) at passage <3 were seeded on 6.5 mm 0.4 μm pore size polyester transwell inserts (Corning Costar Transwell) and differentiated for at least 21 days at air-liquid interface using Air-Liquid Interface Differentiation Medium (LifeLine Cell Technology). Developing HAEs were washed apically at least once per week to remove mucus. Transepithelial/transendothelial resistance (TEER) was measured every 24 hours for 3 days after infection using an EVOM volt/ohm meter coupled with STX2 electrode (World Precision Instruments). For infection, virus (5,000 TCID₅₀ units/transwell) was added apically. Compound was administered to the basolateral chamber, starting with a highest concentration of 0.5 μM 4’-FlU in dose-response assays, and exchanged once daily. Each concentration was tested in three independent replicates (biological repeats). Apically shed virus titers were determined through TCID₅₀ titration on MDCK cells as described above.

Minigenome assay

293T cells were seeded and transfected with 1.0 μg of luciferase-encoding reporter plasmid [26] and 0.5 μg each of PB1, PB2, NP and PA expression plasmids derived from A/California/07/2009 (H1N1), A/Viet Nam/1203/2004 (H5N1), and A/Anhui/1/2013 (H7N9), respectively. Three hours post transfection, 4’-FlU or vehicle (DMSO) volume equivalents were added in 3-fold serial dilution series starting at 20 μM 4’-FlU concentration, followed by incubation for 30 hours at 37°C and determination of luciferase activity using a BioTek Synergy H1 multimode plate reader. Relative activities were calculated according to the formula % relative activity = (RLU_i−RLU_min)/(RLU_max−RLU_min) × 100, with RLU_i specifying sample luciferase value, RLU_min specifying values from equally transfected, vehicle-treated cells that did not receive the PB2 subunit encoding plasmid, and RLU_max specifying values from fully transfected, vehicle-treated cells.

Recombinant IAV RdRP expression and purification

A/CA/07/2009 (H1N1)-derived PA, PB1, and PB2 expression plasmids were subcloned into the pFastbac Dual plasmid expression vector (Invitrogen) with polyhedrin promoter control. An TEV protease-cleavable N-terminal hexahistidine-tag was added to the PA subunit to facilitate purification of the complex through affinity chromatography. The individual plasmids were transformed in DH10bac cells and the resulting bacmid transfected into SF9 cells. After virus recovery, SF9 cells were infected with the three individual viruses in the ratio of 1:1:2. Cells were harvested after 72 hours and lysed in lysis buffer (Tris 50 mM pH 7.4, NaCl 300 mM, NP 40 0.5%, protease inhibitor cocktail, glycerol 10%, TCEP 0.5 mM) on ice for two hours, followed by sonication. Clarified supernatant was incubated at 4°C for 1 hour with Ni-NTA beads which were pre-equilibrated with Tris 50 mM pH 7.4, NaCl 300 mM, NP 40 0.2%, glycerol 10%, TCEP 0.5 mM containing 20 mM imidazole. After extensive washing with Tris 50 mM pH 7.4, NaCl 300 mM, NP 40 0.2%, glycerol 10%, TCEP 0.5 mM containing 20–50 mM imidazole, protein complexes were eluted in buffer Tris 50 mM pH 7.4, NaCl 300 mM, NP 40 0.2%, glycerol 10%, TCEP 0.5 mM containing 250 mM imidazole. Eluted protein fractions were pooled and loaded on heparin HP columns, which were flushed with buffer A (Tris 50 mM pH 7.4, NaCl 250 mM, glycerol 10%), then 0–100% of buffer B (Tris 50 mM pH 7.4, NaCl 1M, glycerol 10%) diluted in buffer A. The polymerase complex peak was observed in ~80% buffer B, which corresponds to approximately 900 mM NaCl in the buffer. Peak fractions were collected separately and dialyzed against buffer C (Tris/Cl 50 mM pH 7.4, NaCl 150 mM, glycerol 10%) in dialyzing cassettes at 4°C overnight. Purified protein complexes were harvested, aliquoted and stored at -80°C until use.

In vitro RdRP primer extension assay

In vitro primer extension assays were carried out essentially as described [24,61]. Briefly, RdRP assay reactions were set up in 20 mM Tris-HCl pH 7.4, 10% glycerol, 1 mM TCEP, 3 mM MnCl2, 2 μM RNA template, 100 μM RNA primer, and nucleotides and 1 μCi of α³²P-labelled ATP (Perkin-Elmer) as specified in the figure legends, followed by incubation at 30°C for 10 minutes. Polymerase complexes were added to the reaction and incubation continued for 1 hour at 30°C. Reactions were stopped with 1 volume of deionized formamide containing 25 mM ethylenediaminetetraacetic acid (EDTA), followed by a denaturation step at 95°C. Amplicons were fractionated on 20% polyacrylamide gels with 7M urea, using a Tris-Borate-EDTA electrophoresis buffer. Amplicons were visualized by autoradiography, using a storage phosphor screen BAS IP MS 2040 E (GE Healthcare Life Sciences) and Typhoon FLA 7000 (GE Healthcare Life Sciences) imager. Densitometry analysis was performed using FIJI 2.1.0. To determine enzyme kinetics (V_max and K_m), Michaelis-Menten equation was applied in Prism 9.4.1 (GraphPad).

Pharmacokinetic property and tissue distribution profiling in mice

Male and female C57BL/6J mice were rested for 3 days prior to study start. Then, animals were gavaged with 1.5 mg/kg bodyweight 4’-FlU in 10 mM sodium citrate with 0.5% Tween 80, final gavage volume 200 μl. Blood samples (150 μl) were taken at 90 minutes, 3 hours and 12 hours after dosing, cleared (2,000 rpm, 5 minutes, 4°C), and extracted plasma stored at -80°C. Organ samples (lung, brain, spleen, heart, kidney and liver) were harvested 12 hours after dosing and snap-frozen in liquid nitrogen. Animal tissues were homogenized with 70% acetonitrile in water that included internal standards. Animal plasma and tissue concentrations of 4’FlU and 4’-FlU-TP were measured by a qualified LC/MS/MS method in MRM mode on a QTRAP 5500 (Sciex, Santa Clara, CA, USA) instrument.

Intranasal infection of mice

Female or male mice (5–8 weeks of age, Balb/c, RAG1 KO or IFNar KO) were purchased from Jackson Laboratories. Upon arrival, mice were rested for at least 3 days, then randomly assigned to study groups and housed under ABSL-2 or ABSL-3 conditions for infections with A/CA/07/2009 (H1N1) recombinants or A/VN12/2003 (H5N1), respectively. Bodyweight was determined twice daily, body temperature determined once daily rectally. For infections, animals were anesthetized with isoflurane or isoflurane/ketamine (H5N1 challenge only), followed by intranasal inoculation with 2.5×10¹ TCID₅₀ units/animal of A/CA/07/2009 (H1N1), 1×10³–1×10⁵ TCID₅₀ units/animal of GFP or nanoLuc expressing reporter virus versions of A/CA/07/2009 (H1N1), or 6×10¹ of A/VN12/2003 (H5N1). In all cases, virus inoculum was diluted in 50 μl and administered in amounts of 25 μl per nare. Animals were euthanized and organs harvested at predefined time points or when animals reached humane study endpoints.

4’-FlU anti-IAV efficacy studies in mice

Mice were inoculated intranasally with IAV stocks as described, followed by treatment with 4’-FlU at specified dose levels and starting time points through oral gavage. Unless specified otherwise, treatment was continued q.d. Each individual study contained animals receiving equal volumes of vehicle through oral gavage. At study end point, lung tissue was harvested for virus titration or histopathology assessment. For tissue fixation, lungs were perfused using 10% neutral-buffered formalin [10% formalin, NaH₂PO₄ (4 g/liter), and Na₂HPO₄ (6.5 g/liter)], dissected, and fixed for 24 hours. Formalin-fixed lungs were transferred to 70% EtOH after 72 hours, embedded in paraffin, sectioned (5-μm thickness), and stained with hematoxylin and eosin. Slides were evaluated by a licensed pathologist. A pathology score range of 0 to 3 was used to evaluate tissue damage. For analysis of proinflammatory cytokine levels through Bioplex, lungs of euthanized mice were lavaged with 1 ml sterile PBS to extract BALFs, and cleared BALF analyzed against the Standard Th17-panel (Bio-Rad) according to the manufacturer’s instructions.

Efficacy studies in ferrets

Female ferrets (6–10 months of age) were purchased from Triple F Farms. Upon arrival, ferrets were rested for 1 week, then randomly assigned to study groups and housed in groups of three animals under ABSL-2 conditions. In some studies, animals received a temperature sensor for continued telemetric measurement of body temperature. Otherwise, body temperature was determined rectally once daily. Dexmedetomidine/ketamine anesthetized animals were inoculated intranasally with 1×10⁵ TCID₅₀ units of A/CA/07/2009 (H1N1) in a volume of 500 μl per nare. Nasal lavages were performed twice daily using 1 ml of PBS containing 2× antibiotics-antimycotics (Gibco). Treatment with 4’-FlU through oral gavage followed a q.d. regimen. Ferrets were terminated four days post infection and BALF and organ (lungs, turbinates and tracheas) samples extracted for virus titrations.

Ferret transmission studies

Female ferrets (3–8 months of age) were anesthetized with dexmedetomidine/ketamine and infected intranasally with 1×10⁵ TCID₅₀ units of A/CA/07/2009 (H1N1) as before. Nasal lavages were performed every 12 hours, bodyweight and temperature determined once daily. Treatment of source animals was initiated 12 or 24 hours after infection at a dose of 2mg/kg bodyweight, and continued q.d. for three doses total. Starting day 2.5 after infection, uninfected and untreated contact animals were added to the infected and treated source ferrets to allow direct-contact transmission, and co-housing continued until termination of the source ferrets on study day 4. Sentinels were subjected to once daily nasal lavages and monitored until study day 10 or, when infectious particles were detected in lavages of contacts of 4’-FlU-treated source animals, study day 14.

Virus titration from tissue samples

Organs were weighed and homogenized in 300 μl PBS using a beat blaster, set to 3 cycles of 30 seconds each at 4°C, separated by 1-minute rest periods. Homogenates were cleared (10 minutes at 20,000 × g and 4°C), and cleared supernatants stored at -80°C until virus titration. Viral titers were expressed as TCID₅₀ units per gram input tissue. For A/VN12/2003 (H5N1), lungs were homogenized using a TissueLyzer (Qiagen), clarified by centrifugation, supernatants aliquoted, and then stored at -80°C. A/VN12/2003 (H5N1) virus titer was determined by plaque assay and expressed as PFU per ml of lung homogenate.

Infection of mice with aerosolized A/CA/07/2009 (H1N1)

An Aeroneb (Kent Scientific) system was used to infect mice (female Balb/c, 6–8 weeks of age) with aerosolized IAV. Conscious animals were transferred to an aerosol chamber placed on a heat pad adjusted to 37°C to prevent condensation, connected to the nebulizer unit (Aeroneb with palladium mesh) and air pump with airflow restrictor. For condition-finding experiments, the nebulizer chamber was loaded with virus inoculum diluted to 1×10³−1×10⁵ TCID₅₀ A/CA/07/2009 (H1N1) per ml in sterile PBS, airflow adjusted to 1 l/minute, and animals continuously exposed for 3, 10, or 30 minutes. For treatment studies, conditions were adjusted to 1×10⁵ TCID₅₀/ml in sterile PBS, 10 minutes exposure time, and 1 l/minute airflow.

Re-infection of 4’-FlU-experienced mice with A/CA/07/2009 (H1N1)

Mice infected with aerosolized A/CA/07/2009 (H1N1) as above were treated q.d. with 4’-FlU or vehicle as specified, and monitored for 10 weeks or until humane endpoints were reached. Blood was collected 4 and 8 weeks after infection and A/CA/07/2009 (H1N1)-neutralizing antibody titers determined. Treated recoverees were reinfected with homotypic, aerosolized A/CA/07/2009 (H1N1) 10 weeks after the original infection along with a fresh set of naïve animals, followed by monitoring for an additional 14 days and final blood collection at study end. No treatment was administered after reinfection.

Neutralizing antibody titers in mice

Plasma was prepared from blood samples (2,000 rpm, 10 minutes, 4°C) and stored at -80°C until analysis. Heat-inactivated plasma was incubated with 100 TCID₅₀ units of A/CA/07/2009 (H1N1) for 90 minutes, followed by serial dilution and transfer to MDCK cells, and plates incubated for 3 days at 37°C. Remaining infectivity was visualized through hemagglutination activity on chicken red blood cells.

Statistical analysis

For statistical analysis of studies consisting of only two groups, unpaired two-tailed t-tests were applied. When comparing more than two study groups, 1-way analysis of variance (ANOVA) or 2-way ANOVA with multiple comparison post hoc tests as specified were used to assess statistical difference between samples. Statistical analyses were carried out in Prism version 9.4.1 (GraphPad). The number of individual biological replicates (n values) and exact P values are shown in the figures when possible. The threshold of statistical significance (α) was set to 0.05. All quantitative source data and statistical analyses are shown in S1 and S2 data files, respectively.