Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice

Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice Elena Govorkova, Konstantin Andreev, Jeremy Jones, Ahmed Kandeil, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7768971/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Intercontinental spread of highly pathogenic avian influenza A(H5N1) viruses poses significant pandemic risks and necessitates strong protective countermeasures. We evaluated the therapeutic efficacy of the neuraminidase inhibitor oseltamivir, the polymerase inhibitors baloxavir and favipiravir, and an ion-channel blocker amantadine, against severe influenza A(H5N1) infection in mice. Oseltamivir (≥ 100 mg/kg/day for 5 days) provided limited survival benefits and failed to prevent viral neuroinvasion. Baloxavir (≥ 10 mg/kg, 1 dose) fully protected mice, significantly reduced virus respiratory replication, and prevented neuroinvasion. Favipiravir (≥ 100 mg/kg/day for 5 days) provided partial protection, with viral titers being reduced in lungs and brain. Amantadine provided no benefits. Although all drugs inhibited A(H5N1) viruses in vitro , in vivo correlations did not extend beyond baloxavir. Our results indicate that baloxavir is the most reliable treatment to address both respiratory replication and systemic spread of contemporary A(H5N1) viruses in mice and should be considered in pandemic planning. Biological sciences/Microbiology/Virology/Influenza virus Biological sciences/Microbiology/Virology/Antivirals Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Influenza viruses are significant human pathogens that cause respiratory disease and annual epidemics 1 . However, they are also infect wild birds, domestic poultry, and other peridomesticated species 2 . This provides opportunities for zoonotic transmission to humans, with some influenza subtypes being capable of causing severe disease and death 3 . One such subtype is A(H5N1). The so-called Goose/Guangdong-lineage A(H5N1) viruses have circulated and genetically diversified into several clades since emerging in southeast Asia in 1996 4 . The recent genetic clade of this lineage, designated clade 2.3.4.4b based on the viral hemagglutinin (HA), has dominated globally and has demonstrated the ability to cause disease in birds, terrestrial mesocarnivores, aquatic mammals, dairy cows, and humans 5 . These viruses are endemic in wild birds in many countries, and the risk of spread to humans directly or via animal intermediates must be countered with epidemiological, biosecurity, and pharmacological control strategies (Updated joint FAO/WHO/WOAH public health assessment of recent influenza A(H5) virus events in animals and people, March 1, 2025). Although no human-to-human transmission of A(H5N1) 2.3.4.4b virus has been reported, 70 human cases of A(H5N1) infection have been documented across 13 U.S. states, mostly linked to direct exposure to infected dairy cattle or poultry ( https://www.cdc.gov/bird-flu/situation-summary/index.html#human-cases , August 1, 2025). Clinical manifestations were generally mild, and mortality rates and public health risks remain low 6 . However severe disease or death in humans after 2.3.4.4b virus infection has been reported in the United States, Canada, Chile, Ecuador, and mostly recently Mexico 7 , 8 (Avian Influenza A(H5N1) - Mexico, April 17, 2025). Severe disease outcomes and neurologic complications are also common in A(H5N1) 2.3.4.4b animal models 9 . Despite efforts to develop novel clade-specific vaccine candidates 10 , 11 , vaccines against circulating clade 2.3.3.4b viruses are not available in large quantities 12 . In cases of established A(H5N1) infection, antivirals may play a pivotal role in clinical management. ( https://www.cdc.gov/bird-flu/treatment/index.html , May 2, 2025). We previously examined the genotypic and phenotypic susceptibility of emerging influenza A(H5N1) viruses (including bovine isolates 13 and those from virus-contaminated raw [unpasteurized] milk 14 to U.S. Food and Drug Administration–approved antivirals in vitro 15 . We found that A(H5N1) 2.3.4.4b viruses from both avian and mammalian species are generally susceptible to the neuraminidase (NA) inhibitor (NAI) class of drugs, which inhibit viral egress, and to the cap-dependent endonuclease inhibitor (CENI) baloxavir, which inhibits the endonuclease activity of the viral acidic polymerase (PA) protein and halts viral gene transcription. We also found that less than 1% of viruses surveyed globally carry markers of reduced susceptibility to NAIs or CENIs 16 . In the United States and many other countries, the NAI oseltamivir (Tamiflu®) is provided as standard-of-care treatment for seasonal influenza and commonly for zoonotic virus infections 17 , whereas baloxavir (Xofluza®) is a new drug with limited usage outside Japan and for which there are few reports of its use to treat zoonotic infections in humans 18 . Two other inhibitors have been associated with influenza treatment. Favipiravir (Avigan®) is a nucleoside-analog polymerase inhibitor targeting the polymerase basic protein 1 (PB1) and thereby causing nucleic acid chain termination or lethal viral mutagenesis 19 . It is approved for use in Japan but with significant restrictions 20 . The adamantane class of drugs, including amantadine (Gorcovi®) target the viral matrix 2 (M2) protein to prevent viral entry, but their use to treat seasonal influenza has been discontinued because of widespread resistance 21 , 22 . However, amantadine and favipiravir have been reconsidered for treating severe A(H5N1) 2.3.4.4b because of their unique mechanisms of action and the ability of amantadine to cross the blood–brain barrier to potentially confront A(H5N1) viral neuroinvasion. In this study, we evaluated the in vivo efficacy of four classes of direct-acting influenza antivirals against lethal and neurotropic A(H5N1) infection in the mouse model. Mice were challenged with two well-characterized avian-origin neurotropic North American clade 2.3.4.4b viruses 9 and received the NAI oseltamivir, the CENI baloxavir acid (the active metabolite of baloxavir), the nucleoside analog favipiravir, or the ion-channel inhibitor amantadine in an effort to understand which—if any—of these drugs could combat these severe infections. RESULTS In vitro antiviral susceptibility of A(H5N1) 2.3.4.4b challenge viruses In vitro phenotypic assays revealed that both A(H5N1) 2.3.4.4b mouse challenge viruses, A/lesser scaup/Georgia/W22-145E/2022 (A/scaup/GA/22) and A/red-shouldered hawk/North Carolina/W22-121/2022 (A/hawk/NC/22), were susceptible to sub- to low-nanomolar concentrations of oseltamivir and baloxavir. The resulting 50% inhibitory/effective concentrations (IC 50 /EC 50 ) were < 10-fold and < 3-fold greater than subtype-matched reference virus values for the NAI and CENI, respectively. This indicated normal drug susceptibility according to current WHO Antiviral Working Group (AVWG) criteria (World Health Organization AVWG Proposed NI Susceptibility Criteria for Surveillance and Reporting). Favipiravir EC 50 values were in the micromolar range, which is comparable to those for pandemic A(H1N1)pdm09 influenza viruses (Table 1 ) and to previously reported data 23 . Amantadine EC 50 values for both viruses were in the sub- to low-micromolar range, similar to those for adamantane-susceptible reference viruses. These values were also < 235- to 2712-fold lower than those for amantadine-resistant A/Vietnam/1203/2004 (H5N1) and A/Illinois/08/2008 (H1N1)pdm09 reference viruses carrying M2-S31N (Table 2 ). Sequence-based analysis of A(H5N1) viruses used for animal studies identified no important clinical amino acid substitutions conferring reduced susceptibility to the four antivirals studied, namely NA-H275Y (for oseltamivir); PA-I38T/F/M/S/L and PA-E23K/G (for baloxavir); PB1-K229R (for favipiravir); and M2- L26F, M2-V27A, M2-A30T/V, M2-S31N, and M2-G34E (for amantadine). Broadly, these data suggest that our mouse challenge viruses have no genetic markers for, or in vitro phenotypes of, reduced drug susceptibility. Pathogenicity of A(H5N1) 2.3.4.4b challenge viruses in mice Control (untreated) mice intranasally inoculated with A/scaup/GA/22 or A/hawk/NC/22 lost weight rapidly, and all succumbed to infection between 5 and 8 days post inoculation (dpi) (Fig. 1–4A & Fig. S1 a,b). The average symptom scores were consistent with morbidity and mortality and peaked progressively at 6 dpi (Fig. 1–4C,D & Fig. S2 ,S3 C, D). More than 60% of control animals (13 of 20) displayed neurologic signs, including hind-limb paresis, ataxia, and tremors. Also characteristic of virus lethality were the short mean survival times (MSTs) of 4.4 days for A/scaup/GA/22 and 5.3 days for A/hawk/NC/22(Fig. 1–4B & Fig. S2 , S3 B). A/scaup/GA/22 titers in lungs of untreated mice were 10 6 –10 7 TCID 50 /mL at 3 and 6 dpi, with brain titers rising from 10 2 to 10 7 TCID 50 /mL between 3 and 6 dpi (Fig. 1–4E, F). Mild to moderate pulmonary lesions and viral nucleoprotein (NP) antigen were distributed throughout 30% of the total lung area by 3 dpi (Fig. S1 c). At 6 dpi and at peak morbidity, the extent of pulmonary lesions increased to 47% and animals exhibited extensive perivascular and peribronchiolar inflammation with bronchiolar epithelial necrosis indicating widespread active infection (Fig. S1 d). Perivascular inflammatory cell infiltrates, gliosis, and vacuolation were present in virus-positive areas of the brainstem, while NP antigen positive cells were found in the olfactory bulb, olfactory cortex, and trigeminal ganglia. Overall, productive A(H5N1) infection was established in the lungs of mice with our two challenge viruses, and both rapidly disseminated to the brain. Oseltamivir efficacy We examined the benefits of pre-exposure prophylaxis and 24 h–delayed treatment with the NAI oseltamivir in mice. Prophylactic oseltamivir initiated at − 4 h post inoculation (hpi) at 100 and 200 mg/kg/day resulted in 40% to 80% survival, MSTs of 12.8 to 17.4 days, initial weight loss of 15% to 10%, and clinical symptoms lasting for 15 to 10 dpi, respectively (Fig. S2 a-d). Oseltamivir administered + 24 hpi had a dose-dependent antiviral effect. Oseltamivir doses of 5 and 10 mg/kg/day did not protect or extend the MST of mice challenged with A/scaup/GA/22 or A/hawk/NC/22 (Fig. S1 ). For A/scaup/GA/22, treatment with 20 mg/kg/day resulted in 0% survival, whereas treatment with 100 or 200 mg/kg/day resulted in 20% and 60% survival, respectively, and MST was extended to 10.4 and 14.4 days, respectively (Fig. 1a,b). Mice treated with 200 mg/kg/day of oseltamivir displayed minimal body weight loss, and survivors recovered weight faster than those treated with 100 mg/kg/day (Fig. 1c,d). Oseltamivir administration did not reduce virus titers in lungs and brain (Fig. 1e,f; Fig. S2 e,f). With oseltamivir treatment at 20 and 200 mg/kg/day, NP antigen–positive cells and pulmonary lesions involved 19% and 13%, respectively, of the lung parenchyma. Lung lesion severity was similar for both regimens (Fig. 1g,h). Overall, oseltamivir treatment with high doses (≥ 100 mg/kg/day) provided partial protection against lethal A(H5N1) infection in mice. Baloxavir efficacy Prophylactic baloxavir initiated at − 4 hpi at 10 mg/kg resulted in 100% survival, MST of ≥ 18 days, absence of weight loss and clinical symptoms, and inhibition of A/scaup/GA/22 replication in the lungs and brain (Fig. S3 a-f). Delayed baloxavir treatment (initiated at + 24 hpi) with 0.1, 1, or 10 mg/kg protected 20%, 40%, and 80% of mice, respectively, with MSTs of 10.2, 13.2, and 17.4 days, respectively (Fig. 2a,b). Delayed treatment with 10 mg/kg prevented weight loss and clinical symptoms (Fig. 2c,d), whereas treatment with ≥ 1 mg/kg significantly reduced viral titers in lungs and brain (Fig. 2e,f). Lungs of mice treated with 0.1 mg/kg were characterized by extensive central pneumonia accompanied by abundant NP antigen (affecting 44% of the lung). In contrast, mice treated with 10 mg/kg showed only multifocal small areas of septal thickening with no detectable NP antigen (Fig. 2g,h). Overall, a single-dose treatment with baloxavir at ≥ 10 mg/kg protected mice from lethal A(H5N1) infection and prevented virus replication in the lungs and brain. Favipiravir efficacy Favipiravir administered at + 24 hpi improved the survival of mice challenged with A/scaup/GA/22 in a dose-dependent manner. Treatment with favipiravir at 20 mg/kg/day resulted in 0% survival, whereas 100 and 300 mg/kg/day resulted in 40% and 60% survival, respectively, and prolonged MSTs of 12.6 and 15.2 days, respectively (Fig. 3a,b). The changes in body weight and clinical scores for mice receiving 100 mg/kg/day and those receiving 300 mg/kg/day did not differ significantly (Fig. 3c,d). Favipiravir treatment at 100 and 300 mg/kg/day doses reduced viral titers in the lungs and brains of mice (Fig. 3e,f). The extent of pulmonary lesions and antigen distribution were markedly reduced in the higher-dose group (Fig. 3g,h). Overall, favipiravir treatment at ≥ 100 mg/kg/day provided partial protection against lethal A(H5N1) infection. Amantadine efficacy Delayed amantadine treatment initiated at + 24 hpi with 15, 30, or 300 mg/kg/day did not improve the survival, MST, weight loss, or clinical score of mice challenged with A/scaup/GA/22 (Fig. 4a-d). This regimen did not reduce the lung and brain viral titers relative to those in untreated control mice (Fig. 4e,f). Importantly, mock-infected mice treated with 300 mg/kg/day of amantadine exhibited significant weight loss and elevated clinical scores indicating possible drug toxicity (Fig. S4 ). Overall, amantadine in all tested treatment regimens failed to protect mice lethally challenged with A(H5N1) clade 2.3.4.4b viruses. Emergence of drug-resistant variants and host adaptation We conducted deep sequencing of 14 paired lung and brain samples from mice that were treated with the highest dosage of each antiviral and of 10 samples from control animals (Table S1 ). We focused on substitutions in the proteins targeted by each drug, e.g., NA, M2, PA, or PB1, with significance being given to substitutions present in ≥ 5% of the viral population and/or that were previously associated with clinical reports of treatment-emergent reduced susceptibility variants. No such substitutions in the NA and PB1 proteins were identified in oseltamivir-treated and favipiravir-treated mice, respectively. One animal treated with amantadine exhibited M2-S31N, the most frequently identified marker of amantadine resistance in seasonal influenza A viruses, as a minor variant (19%). Five previously unreported amino acid substitutions in the N-terminal domain (M2-K12R, M2-N18K, M2-S20N) or C-terminal domain (M2-V51I, M2-G61R) of the M2 ion channel were detected in more than 95% of the viral population in the brain of four amantadine-treated mice. Phenotypic testing of variants carrying these five M2 substitutions did not show reduced susceptibility to amantadine (Table 2 ). PA-I38L, associated with reduced baloxavir susceptibility in vitro 24 , was predominant (present in ≥ 95% of the virus population) in both lung and brain tissues of a single baloxavir-treated mouse. Overall, the NA, PA, and PB1 genetic markers of reduced antiviral susceptibility, including the clinically relevant markers NA-H275Y, NA-E199A/D/G (N1 numbering) (for NAIs), PA-I38T (for baloxavir), and PB1-K229R (for favipiravir) were not identified in drug-treated mice. A portion of variants isolated from both drug-treated and untreated mice carried markers of mammalian host adaptation. PB2-E627K, which enhances viral replication and transmission in mammals 25 , was identified as a minor variant (in 9.6%–32.5% of viruses) in lungs of four of six animals treated with oseltamivir and favipiravir. One animal in the control group had this substitution predominating in both lungs (88%) and brain (≥ 95%) samples. PB2-D701N was found in lung and brain tissues of mice in different treatment groups and in vehicle controls. This substitution within the nuclear localization signal domain of the PB2 protein increases pathogenicity in mice 26 and humans 27 by promoting nuclear import of viral influenza ribonucleoproteins 28 . One animal exhibited minor variants with dual PB2-E627K and PB2-D701N substitutions, which enhance virulence and transmission in mice 29 . The 493–512 region of the PA C-terminal domain is involved in structural interactions with the host transcription factor hCLE, which modulates nuclear RNA metabolism and enhances viral polymerase activity 30 . In this region, we identified numerous instances of PA-C489S, which was particularly predominant in brain tissues (being present in ≥ 95% of viruses). Additionally, PB2-L207I localized within the NP- and PB1-binding sites of the PB2 protein and was identified as a minor variant in lung tissue of oseltamivir- and amantadine-treated animals. It was also predominant in both tissues of two of five control mice. These previously unreported substitutions were considered because of their appearance in multiple animals and treatment groups. DISCUSSION HPAI A(H5N1) clade 2.3.4.4b viruses are characterized by elevated pathogenicity in avian and mammalian species, with potential for virus spread beyond the respiratory tract 31 . Therefore, there is significant concern regarding whether such viruses will cause severe disease in exposed humans and whether the available antivirals can control such infections. In this study, we comprehensively evaluated the potential therapeutic efficacy of four classes of antivirals that target different influenza virus proteins, including those targeting two polymerases and proteins mediating both entry and egress of A(H5N1) clade 2.3.4.4b viruses in mice. Our challenge viruses were the avian-origin A/scaup/GA/22 and A/hawk/NC/22 viruses, which exhibit 100% lethality with viral dissemination in mice 9 . Because the pathogenicity of these viruses is more severe than that of seasonal influenza viruses, we not only evaluated mouse treatment doses approximating human equivalents but also doses 5- to 10-fold greater 32 . Clinical guidance and pharmacokinetic studies were also taken into consideration when selecting the doses 33 – 36 . According to the CDC Interim Guidance on treating novel influenza infections associated with severe human disease ( https://www.cdc.gov/bird-flu/hcp/clinicians-evaluating-patients/interim-guidance-treatment-humans.html , July 3, 2025), treatment longer than 5 days is recommended for hospitalized patients with severe and prolonged illness. A 2-fold greater dose of oseltamivir has been proposed for treating influenza in immunocompromised patients and/or patients exhibiting progressive disease despite early administration of standard-dose medication 37 . NAIs are currently the first-line countermeasures against pandemic influenza 38 , with more than 80% of the U.S. Strategic National Stockpile consisting of orally administered oseltamivir 39 . Oseltamivir IC 50 s of HPAI A(H5N1) clade 2.3.4.4b viruses, including a recent human A/Texas/37/2024 isolate, were in the low-nanomolar range, < 10-fold IC 50 change vs. subtype-matched reference virus values, which aligns with previously reported data and meets the WHO criteria for drug susceptibility 16 . Oseltamivir at 20 mg/kg/day did not protect mice against HPAI A(H5N1) clade 2.3.4.4b virus infection, despite this dose reportedly producing a plasma concentration comparable to that of the recommended human oral dose of 75 mg BID 40 . Increasing the oseltamivir dose 5- to 10-fold provided 20%–60% greater survival. Importantly, oseltamivir did not significantly reduce virus titers in lungs or brains. This may in part be due to the mechanism of NAIs, which primarily target the final step in the viral cycle (budding and egress of the progeny virions), meaning that one or more cycles have been completed in the infected cell(s), along with possible induction of host-cell inflammatory and antiviral responses 41 . In a recent study, oseltamivir (40 mg/kg/day) and zanamivir (8 mg/kg/day) administered orally BID for 5 days were unable to prevent lethality in mice infected with bovine-origin A(H5N1) 2.3.4.4b virus 42 . However, a consistent regimen of baloxavir marboxil (50 mg/kg/day) protected 100% of mice against lethal bovine A(H5N1) infection 42 , although the drug efficacy was significantly reduced when treatment was delayed until 48–72 hpi 43 . Additionally, consistent with previously published data on earlier A(H5N1) viruses 44 , we found no correlation between the survival of mice treated with clinically relevant doses of oseltamivir and in vitro susceptibility. Therefore, for oseltamivir, and possible for other NAI drugs, the in vitro phenotypic susceptibility of A(H5N1) clade 2.3.4.4b viruses may not reliably predict in vivo treatment outcomes. Clinical recommendations for treating sporadic human A(H5N1) infections are primarily based on limited case series of patients and on extrapolation from seasonal influenza practicies 45 . In vivo studies may be considered to include risk assessment practices and pharmacological management of emerging influenza viruses to confirm antiviral efficacy in animal models. Baloxavir was the most successful of the four drugs studied with regard to its capacity to control virus replication in the lungs and brain. The 10 mg/kg dose resulted in 80% survival and significantly reduced virus titer in lungs and brains. This exceeds the 40% survival rate in mice treated with baloxavir marboxil at 50 mg/kg/day BID for 5 days, also starting at + 24 hpi 43 . This may be attributed to differences in the pharmacokinetics of the prodrug and its active metabolite. For critical cases of A(H5N6) infection in humans, baloxavir decreased the viral load and respiratory and serum cytokines, even when treatment was delayed for 3–4 days. However, these data are complicated by the use of adjunct treatments, including NAIs, in these patients, as well as an overall lack of clinical reports of baloxavir use in humans infected with A(H5Nx) viruses 46 . Therefore, preclinical studies remain important potential indicators of drug usage in severe disease cases. Baloxavir targets the PA endonuclease activity, which cleaves capped host pre-mRNAs that can then be used by the virus for its own transcription. As this step occurs earlier in the viral replication cycle than the steps affected by NAIs, targeting it may provide faster arrest of the viral cycle and progeny virus propagation and could play a key role in controlling infections caused by HPAI A(H5N1) viruses, which are characterized by a highly efficient polymerase complex 47 . Favipiravir affects polymerase activity through a mechanism different from that of baloxavir 48 . Favipiravir targets the PB1 protein of the RNA polymerase complex 49 , causing chain termination or lethal mutagenesis and inhibiting viral RNA synthesis slightly downstream of baloxavir but still upstream of NAI inhibition in the viral cycle. Therefore, drugs that act on these steps may provide enhanced benefits similar to those of CENI polymerase inhibitors. However, they currently have limited approval only in Japan because of teratogenicity reported in preclinical animal studies (Pharmaceuticals and Medical Devices Agency. Review report on Avigan, https://www.pmda.go.jp/files/000210319.pdf . Accessed August 25, 2025). Our data suggest that favipiravir provides some survival benefits and limits viral replication in lungs and brain, but it is less effective than baloxavir in terms of reducing morbidity, mortality, and some measures of viral presence in tissues. A(H1N1)pdm09 viruses with the PA-L666F substitution have been isolated from patients after favipiravir administration, 50 although the effect of the substitution on favipiravir susceptibility has not been reported. One virus isolated from a control animal has acquired PB2-E627K, a known virulence-associated marker of mammalian host adaptation 51 . Although seasonal human influenza viruses are largely resistant to amantadine, we included this drug in our study as A(H5N1) viruses recently isolated from humans and birds were susceptible to M2 ion-channel blockers in vitro 52 . Importantly, adamantanes are the only class of influenza antivirals that exhibit saturable transport across the blood–brain barrier 53 , potentially enabling them to control A(H5N1) virus spread within the brain. The frequency of the M2-S31N variant that predominates in most amantadine-resistant seasonal influenza viruses remains low among A(H5N1) strains at < 0.76% (200 of 26,470 strains) (GISAID, Jan 1, 2022 – Aug 15, 2025). Amantadine at 30 mg/kg/day reportedly protected 60% of mice lethally challenged with amantadine-susceptible A/Vietnam/1203/2004 (H5N1) clade 1 virus 54 , and another study showed amantadine pre-exposure prophylaxis to be 100% effective against lethal A(H5N1) infection 55 . In contrast, our study demonstrated the inefficiency of amantadine against currently circulating HPAI A(H5N1) clade 2.3.4.4b viruses, which was not due to the development of drug-resistant variants. Amantadine’s target M2 protein facilitates HA protein fusion of influenza virions with host endosomal membranes by modulating and lowering the pH of the endosome. The fusion pH for the H5 HA protein of bovine-origin influenza A(H5N1) clade 2.3.4.4b viruses is 5.9, outside the pH range associated with human-like receptor binding (pH 5.0–5.5) 56 , although earlier studies found mutations within the fusion peptide pocket of H5 HA that changed the pH of activation 57 . The lower pH of fusion in contemporary A(H5N1) viruses may make them less dependent on M2 ion channel–mediated endosomal pH acidification, which could make amantadine antiviral mechanisms less effective in vivo . However, rapid dissemination of virus into multiple organs during early stages of infection may also limit therapeutic efficacy, particularly with late treatment initiation. Because of the high pathogenicity and neuroinvasiveness of HPAI A(H5N1) viruses 58 , the ability of antivirals to affect early stages of virus replication is crucial to controlling infections by these viruses. Among the four direct-acting antivirals studied, the polymerase inhibitors are the most promising drugs. A previous study demonstrated that the PA and, to a lesser extent, PB2 subunits of the polymerase were responsible for the increased polymerase activity of HPAI A(H5N1) viruses 59 . Baloxavir targets one of these proteins and effectively limited viral titers early in infection/treatment in adolescent and adult outpatients with uncomplicated influenza 60 . Baloxavir was also associated with a significant decline in virus titers and viral RNA load when compared with oseltamivir in Phase III trials 61 . Patients who received baloxavir experienced significantly faster resolution of hypoxia when compared with oseltamivir recipients 62 . In a recent pediatric study in Japan, a single baloxavir dose shortened symptom duration, with early reduction in viral titer, when compared with a 5-day course of oseltamivir 63 . Baloxavir inhibition of the polymerase complex during early stages of infection may prevent the systemic virus spread that is responsible for multiorgan failure and lethality. By contrast, NAIs such as oseltamivir target the last stage of the viral cycle and may be less effective at limiting the initial rounds of viral replication. Our data showed that NAI efficacy was associated with therapy initiation time. Pre-exposure prophylaxis with oseltamivir, beginning at − 4 hpi and using a consistent regimen, improved the survival rate by 20% when compared with delayed treatment (initiated at + 24 hpi). Accelerated failure time modeling showed that a shorter initiation of treatment with oseltamivir reduced illness duration and symptom severity when compared with intervention at 48 h after fever onset 64 . Therapeutic benefits have been found in patients with acute influenza infection when oseltamivir treatment was initiated no later than 36 − 48 h after the onset of symptoms, indicating the importance of early administration 65 . In summary, our study demonstrates that among four classes of influenza antivirals with wide or limited approval, only the polymerase inhibitor baloxavir provides consistent and robust protection and reduction in viral titers after lethal challenge with A(H5N1) clade 2.3.4.4b in mice. The polymerase inhibitor favipiravir provided partial protection against both disease criteria. In contrast, oseltamivir afforded limited survival benefits and did not control viral titers. These results may in part be due to targeting the early steps of the viral replication cycle and may be of significance for A(H5N1) viruses that have increased polymerase activity. For amantadine and oseltamivir, we found little to no correlation between their antiviral efficacy in mice and susceptibility in vitro , confirming previously published data 44 . At present, we encourage prioritization of baloxavir or, potentially, baloxavir drug combinations as a potential frontline therapeutic approach for A(H5N1) clade 2.3.4.4b infections. We also suggest that more prognostic approaches are warranted to evaluate antivirals against emerging influenza viruses. METHODS Ethics statements and biosafety All protocols and procedures were approved by the St. Jude Children’s Research Hospital Institutional Animal Care and Use Committee and complied with the policies of the National Institutes of Health and the Animal Welfare Act. The animal experiments were conducted in an Animal Biosafety Level 3 + containment facility in accordance with the U.S. Federal Select Agent Program regulations (7 CFR Part 331, 9 CFR Part 121.3, 42 CFR Part 73.3). Viruses, cells, and compounds Influenza A(H5N1) viruses were propagated in the allantoic cavities of 10-day-old embryonated chicken eggs at 35°C for up to 48 h. Influenza A(H1N1)pdm09 reference viruses were grown in Madin–Darby canine kidney (MDCK) cells at 37°C for up to 72 h. Aliquots were stored at − 80°C before use. MDCK cells obtained from the American Type Culture Collection were grown in culture in Modified Eagle’s Medium (MEM) (Thermo Fisher) supplemented with 5% fetal bovine serum (HyClone), 1 mM L-glutamine, and 1× penicillin/streptomycin/amphotericin B (Gibco). Oseltamivir carboxylate and phosphate, baloxavir acid, and favipiravir were purchased from MedChem Express, amantadine was purchased from Sigma-Aldrich. Oseltamivir carboxylate/phosphate and amantadine were resuspended in ultrapure sterile water; stock solutions for baloxavir acid and favipiravir were prepared in sterile DMSO. NAI susceptibility Phenotypic susceptibility to oseltamivir carboxylate was determined by a fluorescence-based assay measuring the ability of the NA protein to cleave 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA) substrate 66 . The NA activity of tested influenza viruses was standardized to relative fluorescent units equivalent to 10 µM 4-methylumbelliferone sodium salt (4-MU) (Sigma-Aldrich), and the viruses were incubated with oseltamivir carboxylate (5 pM to 50 µM) at 37°C for 30 min. The fluorescence signal from the NA-cleaved fluorogenic MUNANA substrate was measured at excitation/emission wavelengths of 360/460 nm. Half-maximal inhibitory concentrations (IC 50s ) were estimated from dose–response curves by using a sigmoidal, four-parameter logistic nonlinear regression equation (in GraphPad Prism v.10.1.2). CENI susceptibility Phenotypic susceptibility to baloxavir was determined by an NA-based replication inhibition assay in MDCK cells 67 . Virus inoculum was normalized to 1.9 nM/well of 4-MU and was incubated with baloxavir (6 pM to 111 nM) on MDCK cells at 37°C for 12 h without TPCK-treated trypsin. The NA activity of the cell-attached virus was measured as an indicator of PA activity, and baloxavir half-maximal effective concentrations (EC 50s ) were calculated as described above. Favipiravir and amantadine susceptibility Phenotypic susceptibility to favipiravir and amantadine was determined by plaque-reduction assays. MDCK cells were inoculated with virus at a dose yielding 50–100 plaque-forming units (PFUs). After incubation, cell monolayers were washed and overlaid with 0.45% immunodiffusion-grade agarose (MP Biomedical) in DMEM with 4% bovine serum albumin, 1 µg/mL TPCK-treated trypsin (Worthington Biochemical) (for non-A(H5N1) viruses), and 10-fold dilutions of favipiravir/amantadine (1 nM to 1 mM). For amantadine, the inoculum was pre-incubated with amantadine at corresponding concentrations at 37°C for 1 h. At 48–72 hpi, cells were fixed with 10% formaldehyde and stained with 1% crystal violet, and the PFUs per well were enumerated. EC 50 s were determined by using the log (inhibitor) versus response logistic nonlinear regression equation. Antiviral efficacy in mice Female 6-week-old BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were lightly anesthetized with isoflurane and inoculated intranasally with 5 × 50% mouse lethal doses (MLD 50 s) of each virus in 50 µL of PBS. Starting at 24 hpi, mice (n = 5/group) were treated by oral gavage (100 µL/mouse) BID (at 12 h intervals) for 5 days with 20, 100, or 200 mg/kg/day of oseltamivir; 20, 100, or 300 mg/kg/day of favipiravir; or 30 or 300 mg/kg/day of amantadine. Baloxavir acid was administered subcutaneously as a single dose at 0.1, 1, or 10 mg/kg (100 µL/mouse). Control animals (n = 5/group) received 100 µL of vehicle (ORA-Plus ® /water) orally BID for 5 days. For the prophylactic regimens, administration of oseltamivir or baloxavir was initiated at − 4 hpi. Mice were weighed daily and monitored for morbidity and mortality (death or loss of ≥ 25% of their initial body weight) for 21 dpi (Fig. S5 ). A daily clinical score for each group was based on the clinical signs of disease, such as lethargy, ruffled fur, hunched posture, respiratory distress (labored breathing), and neurologic complications. MST was calculated by the Kaplan–Meier method 68 . For tissue titration, mice (n = 3/group) were euthanized at 3 and 6 dpi, and the brains and lungs were collected and homogenized with a TissueLyser system (Qiagen) in 1 mL of MEM. The homogenates were cleared by centrifugation at 3000 × g for 15 min and used to determine 50% tissue culture infectious doses (TCID 50 s) by inoculating log10 dilutions onto MDCK cells (with incubation at 37°C for 3 days). TCID 50 values were determined by the method of Reed and Muench 69 . Histopathology and immunohistochemistry At 3 and 6 dpi, mice (n = 3/group) were euthanized, and the lungs and nasal turbinates were infused via the trachea with 10% neutral-buffered formalin (NBF) (Thermo Fisher). The lungs and brains were then collected and fixed by immersion in 10% NBF before being embedded, sectioned, and stained with hematoxylin and eosin (HE) or subjected to immunohistochemical labeling with a rabbit monoclonal antibody/to influenza A virus nucleoprotein (GTX636247, GeneTex, Irvine, CA). Slides were scanned and the percentage areas of lung parenchyma classified as normal versus virus-positive/inflamed were determined by quantitative morphometry using the HALO™ MiniNet AI algorithm (IndicaLabs, Albuquerque, NM). Sequencing Viral RNA was isolated from mouse lung and brain homogenates with an RNeasy Kit (Qiagen). PA, NA, M2, PB1 and PB2 gene segments were amplified from total RNA with universal or gene-specific primers and purified with a QIAquick PCR Purification Kit (Qiagen). Libraries were prepared with a Nextera XT DNA Library Prep Kit (Illumina) and sequenced with a MiSeq Reagent Kit v2 (300 cycles) on a MiSeq System (Illumina). Reads were quality trimmed and assembled with CLC Genomics Workbench v24 (Qiagen). Amino acid variants were called with a variant frequency cutoff of 5% for ≥ 1000 reads. Consensus sequences were aligned using BioEdit v7.0.9 software 70 . Data analysis was based on NA and PA molecular markers associated with reduced or highly reduced inhibition by NAIs or the CENI as summarized by the World Health Organization Antiviral Working Group (Summary of NA amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors; (PA) amino acid substitutions analyzed for their effects on baloxavir susceptibility). PB1-K229R was monitored for resistance to favipiravir 71 . M2 substitutions at residues 26, 27, 30, 31, and 34 were monitored for resistance to amantadine. Serology Post-infection mouse serum samples were obtained at 21 dpi, treated with a receptor-destroying enzyme (Denka), heat inactivated at 56°C for 1 h, and tested for the presence of anti-HA antibodies by performing HA inhibition assays with chicken red blood cells. The reciprocal of the last serum dilution that inhibited hemagglutination was recorded as the HI titer (Fig. S6 ). Statistical analysis Data were analyzed by GraphPad Prism 10.1.2 software (La Jolla, CA), with individual significance being determined by unpaired t- tests and/or one-way analysis of variance (ANOVA). Table 1 Susceptibility of A(H5N1) viruses to the NAI oseltamivir carboxylate, the CENI baloxavir, and the RNA-dependent RNA polymerase inhibitor favipiravir. Influenza A virus IC 50 /EC 50 ± SD a Oseltamivir [nM] Baloxavir [nM] T-705 [µM] A(H5N1) viruses A/red-shouldered hawk/North Carolina/W22-121/2022 3.15 ± 0.53 0.25 ± 0.01 3.99 ± 0.17 A/lesser scaup/Georgia/W22-145E/2022 2.57 ± 0.29 0.31 ± 0.16 6.75 ± 0.88 A/Texas/37/2024 3.19 ± 0.72 0.23 ± 0.07 12.70 ± 0.07 A/Vietnam/1203/2004 0.12 ± 0.00 0.25 ± 0.13 11.46 ± 0.60 A(H1N1)pdm09 reference viruses A/Denmark/528/2009 NA-H275Y b 137.33 ± 0.25 0.72 ± 0.19 2.40 ± 0.19 A/Illinois/08/2018 0.23 ± 0.00 0.66 ± 0.07 3.45 ± 0.64 A/Illinois/308/2018 PA-I38T b 0.25 ± 0.04 34.53 ± 16.92 1.91 ± 1.00 a The results are representative of two or three independent dose–response curves ± the standard deviation (SD). b N1 numbering. NA/PA substitutions that conferred reduced inhibition/highly reduced inhibition by oseltamivir/baloxavir (IC 50 /EC 50 fold-change ≥ 10/>3) are indicated in bold. Declarations Competing interests: The authors declare no competing interests. Data and materials availability All data are included in manuscript figures and text. Funding: This project was funded by the U.S. NIAID, U.S. NIH, and U.S. DHHS under contract 75N93021C00016. This content is the responsibility of the authors and does not necessarily represent official views of the U.S. NIH. Author contributions: KA, RJW and EAG conceptualized the research project. KA, JCJ, AK, PV acquired and/or analyzed the data. KA, JCJ and EAG wrote original drafts, and all authors reviewed and edited subsequent drafts. Funding was acquired by RJW. ACKNOWLEDGMENTS: The authors thank Keith A. Laycock, PhD, ELS, for scientific editing of the manuscript, David Carey, Heather Weinberg, and Chelsi Stultz for animal husbandry, the St. Jude Hartwell Center for Bioinformatics and Biotechnology for next-generation sequencing, Patrick Seiler, Jeri-Carol Crumpton for experimental assistance. 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Oxford University Press, pp. 95–98 Goldhill DH et al (2018), The mechanism of resistance to favipiravir in influenza. Proc. Natl. Acad. Sci. U. S. A. 115, 11613–11618 Additional Declarations There is NO Competing Interest. Supplementary Files AndreevNatureMedicineSuppInfo.docx Supplemental Material FigureS2.pdf Supplemental Figure S2 FigureS6.pdf Supplemental Figure S6 FigureS3.pdf Supplemental Figure S3 FigureS4.pdf Supplemental Figure S4 FigureS1.pdf Supplemental Figure S1 FigureS5.pdf Supplemental Figure S5 Cite Share Download PDF Status: Published Journal Publication published 19 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7768971","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":527310042,"identity":"f3a7b543-c1eb-47ad-b7d1-6a88a77f47b1","order_by":0,"name":"Elena Govorkova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYBAC+3YwZQcimEGEnPz8h8+kP/5hkLNvP4BViwFYHUMyXIuxwWGGNGnJBiDjTAI+LQfgWhI3MAO18DYAGTewO8yAmfnYgx8MB+QMjvc+NvjYZpdOUIs9M1u6YQ/DAaAzjhsnzmxLzp3fDNLygyFx/wxctvCYSfAwHE+cOSON+TDPGebchsNQW/ZJPMChhf+b5B+GwzAt9ekMMC2bJXB5n4dNmgeopV8ijTmZp+JwAsNhFoiWjTNwaWEzk5YxSDbm5znGbDij4rjhhsNsh40lGyRwBrJ9e/MzyTcVdnJs7G3MEh8MquXlm5kbH3/8YwMMwwPY/Q+xC1NIAo/yUTAKRsEoGAWEAACIk1sLNh7A5wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9067-5682","institution":"St. Jude Children's Research Hospital","correspondingAuthor":true,"prefix":"","firstName":"Elena","middleName":"","lastName":"Govorkova","suffix":""},{"id":527310043,"identity":"ae5a9c0a-b1d7-4368-9bea-0efeff4888ff","order_by":1,"name":"Konstantin Andreev","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Andreev","suffix":""},{"id":527310044,"identity":"9494291f-3981-4b11-928c-b2c3653dc5ae","order_by":2,"name":"Jeremy Jones","email":"","orcid":"https://orcid.org/0000-0002-9980-2112","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Jones","suffix":""},{"id":527310045,"identity":"97bdc760-285e-4795-a8ee-442714cdd575","order_by":3,"name":"Ahmed Kandeil","email":"","orcid":"https://orcid.org/0000-0003-3253-6961","institution":"National Research Center","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"","lastName":"Kandeil","suffix":""},{"id":527310046,"identity":"faff8b3b-423d-436c-aa0d-94623abcc11b","order_by":4,"name":"Peter Vogel","email":"","orcid":"https://orcid.org/0000-0002-7535-0545","institution":"St Judes Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Vogel","suffix":""},{"id":527310047,"identity":"af5e7eac-b83c-4030-b173-931a37ce67b3","order_by":5,"name":"Richard Webby","email":"","orcid":"https://orcid.org/0000-0002-4397-7132","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Webby","suffix":""}],"badges":[],"createdAt":"2025-10-02 20:46:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7768971/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7768971/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69721-5","type":"published","date":"2026-02-19T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":93950391,"identity":"47f04ac1-a562-4ce6-8fcd-8f814f94de5e","added_by":"auto","created_at":"2025-10-20 14:54:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":470898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDelayed oseltamivir treatment of mice challenged with A/scaup/GA/22. a–f, \u003c/strong\u003eSurvival (\u003cstrong\u003ea\u003c/strong\u003e), mean survival time (MST) (\u003cstrong\u003eb\u003c/strong\u003e), body weight (\u003cstrong\u003ec\u003c/strong\u003e), clinical score (\u003cstrong\u003ed\u003c/strong\u003e), and virus titers in lung tissue (\u003cstrong\u003ee\u003c/strong\u003e) and brain tissue (\u003cstrong\u003ef\u003c/strong\u003e) at 3 dpi and 6 dpi in BALB/c mice challenged with 5 MLD\u003csub\u003e50 \u003c/sub\u003eof A/scaup/GA/22 and treated with oseltamivir (oral) starting at +24 hpi. \u003cstrong\u003eg,h,\u003c/strong\u003e Histopathologic changes in lungs of infected mice treated with 20 mg/kg/day (\u003cstrong\u003eg\u003c/strong\u003e) or 200 mg/kg/day (\u003cstrong\u003eh\u003c/strong\u003e) of oseltamivir. A representative section is shown for each antiviral dose. The total lung areas examined are outlined in green; red-shaded areas are bronchioles/alveoli with active virus infection (i.e., containing antigen-positive cells). The average percentage area with active virus infection is shown in red text, and the average severity score ± the standard error of the mean (SEM) for lung lesions and inflammation is shown in blue text. ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05; **\u003cem\u003eP \u003c/em\u003e≤ 0.01. L.O.D., lower limit of virus detection (10 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL).\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/603c8da23b0379921ba4ae5d.jpg"},{"id":93950859,"identity":"df7b29c7-4aba-40bd-8a08-f0a4d569284b","added_by":"auto","created_at":"2025-10-20 15:02:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDelayed baloxavir treatment of mice challenged with A/scaup/GA/22. a–f, \u003c/strong\u003eSurvival (\u003cstrong\u003ea\u003c/strong\u003e), mean survival time (MST) (\u003cstrong\u003eb\u003c/strong\u003e), body weight (\u003cstrong\u003ec\u003c/strong\u003e), clinical score (\u003cstrong\u003ed\u003c/strong\u003e), and virus titers in lung tissue (\u003cstrong\u003ee\u003c/strong\u003e) and brain tissue (\u003cstrong\u003ef\u003c/strong\u003e) at 3 dpi and 6 dpi in BALB/c mice challenged with 5 MLD\u003csub\u003e50 \u003c/sub\u003eof A/scaup/GA/22 and treated with baloxavir (subcutaneous) starting at +24 hpi. \u003cstrong\u003eg,h,\u003c/strong\u003e Histopathologic changes in lungs of infected mice treated with 0.1 mg/kg (\u003cstrong\u003eg\u003c/strong\u003e) or 10 mg/kg (\u003cstrong\u003eh\u003c/strong\u003e) of baloxavir. A representative section is shown for each antiviral dose. The total lung areas examined are outlined in green; red-shaded areas are bronchioles/alveoli with active virus infection (i.e., containing antigen-positive cells). The average percentage area with active virus infection is shown in red text, and the average severity score ± SEM for lung lesions and inflammation is shown in blue text. ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05; *\u003cem\u003eP \u003c/em\u003e≤ 0.05; **\u003cem\u003eP \u003c/em\u003e≤ 0.01; ***\u003cem\u003eP \u003c/em\u003e≤ 0.001. L.O.D., lower limit of virus detection (10 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL).\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/a9ebffa5bf46da47e0b4cc0c.jpg"},{"id":93950386,"identity":"767b0e3b-e536-41c1-8261-d903a40d4caf","added_by":"auto","created_at":"2025-10-20 14:54:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFavipiravir treatment of mice challenged with A/scaup/GA/22. a–f, \u003c/strong\u003eSurvival (\u003cstrong\u003ea\u003c/strong\u003e), mean survival time (MST) (\u003cstrong\u003eb\u003c/strong\u003e), body weight (\u003cstrong\u003ec\u003c/strong\u003e), clinical score (\u003cstrong\u003ed\u003c/strong\u003e), and virus titers in lung tissue (\u003cstrong\u003ee\u003c/strong\u003e) and brain tissue (\u003cstrong\u003ef\u003c/strong\u003e) at 6 dpi in BALB/c mice challenged with 5 MLD\u003csub\u003e50 \u003c/sub\u003eof A/scaup/GA/22 and treated with favipiravir (oral) starting at +24 hpi. \u003cstrong\u003eg,h,\u003c/strong\u003e Histopathologic changes in lungs of infected mice treated with 20 mg/kg/day (\u003cstrong\u003eg\u003c/strong\u003e) or 300 mg/kg/day (\u003cstrong\u003eh\u003c/strong\u003e) of favipiravir. A representative section is shown for each antiviral dose. The total lung areas examined are outlined in green; red-shaded areas are bronchioles/alveoli with active virus infection (i.e., containing antigen-positive cells). The average percentage area with active virus infection is indicated in red text, and the average severity score ± standard SEM for lung lesions and inflammation is shown in blue text. ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05; *\u003cem\u003eP \u003c/em\u003e≤ 0.05; ***\u003cem\u003eP \u003c/em\u003e≤ 0.001. L.O.D., lower limit of virus detection (10 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL).\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/f5e0e32e7c21768958334f00.jpg"},{"id":93950855,"identity":"0ca0de64-6b3e-44b1-85c9-efd428ed91d9","added_by":"auto","created_at":"2025-10-20 15:02:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":287975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmantadine treatment of mice challenged with A/scaup/GA/22. a–d, \u003c/strong\u003eSurvival (\u003cstrong\u003ea\u003c/strong\u003e), mean survival time (MST) (\u003cstrong\u003eb\u003c/strong\u003e), body weight (\u003cstrong\u003ec\u003c/strong\u003e), clinical score (\u003cstrong\u003ed\u003c/strong\u003e), and virus titers in lung tissue (\u003cstrong\u003ee\u003c/strong\u003e) and brain tissue (\u003cstrong\u003ef\u003c/strong\u003e) at 6 dpi in BALB/c mice challenged with 5 MLD\u003csub\u003e50 \u003c/sub\u003eof A/scaup/GA/22 and treated with amantadine (oral) starting at +24 hpi. ns, \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05; L.O.D., lower limit of virus detection (10 log\u003csub\u003e10\u003c/sub\u003eTCID\u003csub\u003e50\u003c/sub\u003e/mL).\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/bdcf63307ac9bfc487942f2d.jpg"},{"id":105618282,"identity":"f479ffe4-6d98-4feb-9fac-0105632fd9b6","added_by":"auto","created_at":"2026-03-28 07:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2674570,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/d5bf5675-a19a-4a27-b712-9e223e528221.pdf"},{"id":93950849,"identity":"fcba50c3-498e-47b9-8d36-fb8c982c64c1","added_by":"auto","created_at":"2025-10-20 15:02:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":81183,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"AndreevNatureMedicineSuppInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/e62f2b488d440db340779b4d.docx"},{"id":93950387,"identity":"95bf958c-db46-4916-9fff-8fffeb178827","added_by":"auto","created_at":"2025-10-20 14:54:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28005,"visible":true,"origin":"","legend":"Supplemental Figure S2","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/3dffac8aab7c953225ac2f18.pdf"},{"id":93950396,"identity":"953b021e-de42-4fd3-abcf-01788488c706","added_by":"auto","created_at":"2025-10-20 14:54:07","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9412,"visible":true,"origin":"","legend":"Supplemental Figure S6","description":"","filename":"FigureS6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/bef479a296e8dc767a234364.pdf"},{"id":93950385,"identity":"63caf133-e846-4f3b-8ff4-0371b414845c","added_by":"auto","created_at":"2025-10-20 14:54:05","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27326,"visible":true,"origin":"","legend":"Supplemental Figure S3","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/d961f42b700425cac7f88942.pdf"},{"id":93951741,"identity":"51ba3739-7334-4e65-9154-b4f6e4081ff1","added_by":"auto","created_at":"2025-10-20 15:10:06","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16603,"visible":true,"origin":"","legend":"Supplemental Figure S4","description":"","filename":"FigureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/3b143d0814700b10e28961ed.pdf"},{"id":93950390,"identity":"34ac9773-14b9-499a-baed-42af41676d91","added_by":"auto","created_at":"2025-10-20 14:54:06","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1852421,"visible":true,"origin":"","legend":"Supplemental Figure S1","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/fc294f6c332f012372f781b6.pdf"},{"id":93950394,"identity":"d163cc08-2c55-40ec-8743-74797f6d674b","added_by":"auto","created_at":"2025-10-20 14:54:06","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14448063,"visible":true,"origin":"","legend":"Supplemental Figure S5","description":"","filename":"FigureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7768971/v1/8bb92852bbb7848b97385274.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eInfluenza viruses are significant human pathogens that cause respiratory disease and annual epidemics\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, they are also infect wild birds, domestic poultry, and other peridomesticated species\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This provides opportunities for zoonotic transmission to humans, with some influenza subtypes being capable of causing severe disease and death\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. One such subtype is A(H5N1). The so-called Goose/Guangdong-lineage A(H5N1) viruses have circulated and genetically diversified into several clades since emerging in southeast Asia in 1996\u003csup\u003e4\u003c/sup\u003e. The recent genetic clade of this lineage, designated clade 2.3.4.4b based on the viral hemagglutinin (HA), has dominated globally and has demonstrated the ability to cause disease in birds, terrestrial mesocarnivores, aquatic mammals, dairy cows, and humans\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These viruses are endemic in wild birds in many countries, and the risk of spread to humans directly or via animal intermediates must be countered with epidemiological, biosecurity, and pharmacological control strategies (Updated joint FAO/WHO/WOAH public health assessment of recent influenza A(H5) virus events in animals and people, March 1, 2025). Although no human-to-human transmission of A(H5N1) 2.3.4.4b virus has been reported, 70 human cases of A(H5N1) infection have been documented across 13 U.S. states, mostly linked to direct exposure to infected dairy cattle or poultry (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/bird-flu/situation-summary/index.html#human-cases\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/bird-flu/situation-summary/index.html#human-cases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, August 1, 2025). Clinical manifestations were generally mild, and mortality rates and public health risks remain low\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However severe disease or death in humans after 2.3.4.4b virus infection has been reported in the United States, Canada, Chile, Ecuador, and mostly recently Mexico\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (Avian Influenza A(H5N1) - Mexico, April 17, 2025). Severe disease outcomes and neurologic complications are also common in A(H5N1) 2.3.4.4b animal models\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite efforts to develop novel clade-specific vaccine candidates\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, vaccines against circulating clade 2.3.3.4b viruses are not available in large quantities\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In cases of established A(H5N1) infection, antivirals may play a pivotal role in clinical management. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/bird-flu/treatment/index.html\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/bird-flu/treatment/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, May 2, 2025). We previously examined the genotypic and phenotypic susceptibility of emerging influenza A(H5N1) viruses (including bovine isolates\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and those from virus-contaminated raw [unpasteurized] milk\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e to U.S. Food and Drug Administration\u0026ndash;approved antivirals \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. We found that A(H5N1) 2.3.4.4b viruses from both avian and mammalian species are generally susceptible to the neuraminidase (NA) inhibitor (NAI) class of drugs, which inhibit viral egress, and to the cap-dependent endonuclease inhibitor (CENI) baloxavir, which inhibits the endonuclease activity of the viral acidic polymerase (PA) protein and halts viral gene transcription. We also found that less than 1% of viruses surveyed globally carry markers of reduced susceptibility to NAIs or CENIs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the United States and many other countries, the NAI oseltamivir (Tamiflu\u0026reg;) is provided as standard-of-care treatment for seasonal influenza and commonly for zoonotic virus infections\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, whereas baloxavir (Xofluza\u0026reg;) is a new drug with limited usage outside Japan and for which there are few reports of its use to treat zoonotic infections in humans\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTwo other inhibitors have been associated with influenza treatment. Favipiravir (Avigan\u0026reg;) is a nucleoside-analog polymerase inhibitor targeting the polymerase basic protein 1 (PB1) and thereby causing nucleic acid chain termination or lethal viral mutagenesis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. It is approved for use in Japan but with significant restrictions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The adamantane class of drugs, including amantadine (Gorcovi\u0026reg;) target the viral matrix 2 (M2) protein to prevent viral entry, but their use to treat seasonal influenza has been discontinued because of widespread resistance\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, amantadine and favipiravir have been reconsidered for treating severe A(H5N1) 2.3.4.4b because of their unique mechanisms of action and the ability of amantadine to cross the blood\u0026ndash;brain barrier to potentially confront A(H5N1) viral neuroinvasion.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the \u003cem\u003ein vivo\u003c/em\u003e efficacy of four classes of direct-acting influenza antivirals against lethal and neurotropic A(H5N1) infection in the mouse model. Mice were challenged with two well-characterized avian-origin neurotropic North American clade 2.3.4.4b viruses\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and received the NAI oseltamivir, the CENI baloxavir acid (the active metabolite of baloxavir), the nucleoside analog favipiravir, or the ion-channel inhibitor amantadine in an effort to understand which\u0026mdash;if any\u0026mdash;of these drugs could combat these severe infections.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantiviral susceptibility of A(H5N1) 2.3.4.4b challenge viruses\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e phenotypic assays revealed that both A(H5N1) 2.3.4.4b mouse challenge viruses, A/lesser scaup/Georgia/W22-145E/2022 (A/scaup/GA/22) and A/red-shouldered hawk/North Carolina/W22-121/2022 (A/hawk/NC/22), were susceptible to sub- to low-nanomolar concentrations of oseltamivir and baloxavir. The resulting 50% inhibitory/effective concentrations (IC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e) were \u0026lt;\u0026thinsp;10-fold and \u0026lt;\u0026thinsp;3-fold greater than subtype-matched reference virus values for the NAI and CENI, respectively. This indicated normal drug susceptibility according to current WHO Antiviral Working Group (AVWG) criteria (World Health Organization AVWG Proposed NI Susceptibility Criteria for Surveillance and Reporting). Favipiravir EC\u003csub\u003e50\u003c/sub\u003e values were in the micromolar range, which is comparable to those for pandemic A(H1N1)pdm09 influenza viruses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and to previously reported data\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Amantadine EC\u003csub\u003e50\u003c/sub\u003e values for both viruses were in the sub- to low-micromolar range, similar to those for adamantane-susceptible reference viruses. These values were also \u0026lt;\u0026thinsp;235- to 2712-fold lower than those for amantadine-resistant A/Vietnam/1203/2004 (H5N1) and A/Illinois/08/2008 (H1N1)pdm09 reference viruses carrying M2-S31N (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSequence-based analysis of A(H5N1) viruses used for animal studies identified no important clinical amino acid substitutions conferring reduced susceptibility to the four antivirals studied, namely NA-H275Y (for oseltamivir); PA-I38T/F/M/S/L and PA-E23K/G (for baloxavir); PB1-K229R (for favipiravir); and M2- L26F, M2-V27A, M2-A30T/V, M2-S31N, and M2-G34E (for amantadine). Broadly, these data suggest that our mouse challenge viruses have no genetic markers for, or \u003cem\u003ein vitro\u003c/em\u003e phenotypes of, reduced drug susceptibility.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePathogenicity of A(H5N1) 2.3.4.4b challenge viruses in mice\u003c/h2\u003e\u003cp\u003eControl (untreated) mice intranasally inoculated with A/scaup/GA/22 or A/hawk/NC/22 lost weight rapidly, and all succumbed to infection between 5 and 8 days post inoculation (dpi) (Fig.\u0026nbsp;1\u0026ndash;4A \u0026amp; Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea,b). The average symptom scores were consistent with morbidity and mortality and peaked progressively at 6 dpi (Fig.\u0026nbsp;1\u0026ndash;4C,D \u0026amp; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e,S3 C, D). More than 60% of control animals (13 of 20) displayed neurologic signs, including hind-limb paresis, ataxia, and tremors. Also characteristic of virus lethality were the short mean survival times (MSTs) of 4.4 days for A/scaup/GA/22 and 5.3 days for A/hawk/NC/22(Fig.\u0026nbsp;1\u0026ndash;4B \u0026amp; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S3 B).\u003c/p\u003e\u003cp\u003eA/scaup/GA/22 titers in lungs of untreated mice were 10\u003csup\u003e6\u003c/sup\u003e\u0026ndash;10\u003csup\u003e7\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL at 3 and 6 dpi, with brain titers rising from 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e7\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL between 3 and 6 dpi (Fig.\u0026nbsp;1\u0026ndash;4E, F). Mild to moderate pulmonary lesions and viral nucleoprotein (NP) antigen were distributed throughout 30% of the total lung area by 3 dpi (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). At 6 dpi and at peak morbidity, the extent of pulmonary lesions increased to 47% and animals exhibited extensive perivascular and peribronchiolar inflammation with bronchiolar epithelial necrosis indicating widespread active infection (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed). Perivascular inflammatory cell infiltrates, gliosis, and vacuolation were present in virus-positive areas of the brainstem, while NP antigen positive cells were found in the olfactory bulb, olfactory cortex, and trigeminal ganglia. Overall, productive A(H5N1) infection was established in the lungs of mice with our two challenge viruses, and both rapidly disseminated to the brain.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOseltamivir efficacy\u003c/h3\u003e\n\u003cp\u003eWe examined the benefits of pre-exposure prophylaxis and 24 h\u0026ndash;delayed treatment with the NAI oseltamivir in mice. Prophylactic oseltamivir initiated at \u0026minus;\u0026thinsp;4 h post inoculation (hpi) at 100 and 200 mg/kg/day resulted in 40% to 80% survival, MSTs of 12.8 to 17.4 days, initial weight loss of 15% to 10%, and clinical symptoms lasting for 15 to 10 dpi, respectively (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea-d). Oseltamivir administered\u0026thinsp;+\u0026thinsp;24 hpi had a dose-dependent antiviral effect. Oseltamivir doses of 5 and 10 mg/kg/day did not protect or extend the MST of mice challenged with A/scaup/GA/22 or A/hawk/NC/22 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For A/scaup/GA/22, treatment with 20 mg/kg/day resulted in 0% survival, whereas treatment with 100 or 200 mg/kg/day resulted in 20% and 60% survival, respectively, and MST was extended to 10.4 and 14.4 days, respectively (Fig.\u0026nbsp;1a,b). Mice treated with 200 mg/kg/day of oseltamivir displayed minimal body weight loss, and survivors recovered weight faster than those treated with 100 mg/kg/day (Fig.\u0026nbsp;1c,d). Oseltamivir administration did not reduce virus titers in lungs and brain (Fig.\u0026nbsp;1e,f; Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee,f). With oseltamivir treatment at 20 and 200 mg/kg/day, NP antigen\u0026ndash;positive cells and pulmonary lesions involved 19% and 13%, respectively, of the lung parenchyma. Lung lesion severity was similar for both regimens (Fig.\u0026nbsp;1g,h). Overall, oseltamivir treatment with high doses (\u0026ge;\u0026thinsp;100 mg/kg/day) provided partial protection against lethal A(H5N1) infection in mice.\u003c/p\u003e\n\u003ch3\u003eBaloxavir efficacy\u003c/h3\u003e\n\u003cp\u003eProphylactic baloxavir initiated at \u0026minus;\u0026thinsp;4 hpi at 10 mg/kg resulted in 100% survival, MST of \u0026ge;\u0026thinsp;18 days, absence of weight loss and clinical symptoms, and inhibition of A/scaup/GA/22 replication in the lungs and brain (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea-f). Delayed baloxavir treatment (initiated at +\u0026thinsp;24 hpi) with 0.1, 1, or 10 mg/kg protected 20%, 40%, and 80% of mice, respectively, with MSTs of 10.2, 13.2, and 17.4 days, respectively (Fig.\u0026nbsp;2a,b). Delayed treatment with 10 mg/kg prevented weight loss and clinical symptoms (Fig.\u0026nbsp;2c,d), whereas treatment with \u0026ge;\u0026thinsp;1 mg/kg significantly reduced viral titers in lungs and brain (Fig.\u0026nbsp;2e,f). Lungs of mice treated with 0.1 mg/kg were characterized by extensive central pneumonia accompanied by abundant NP antigen (affecting 44% of the lung). In contrast, mice treated with 10 mg/kg showed only multifocal small areas of septal thickening with no detectable NP antigen (Fig.\u0026nbsp;2g,h). Overall, a single-dose treatment with baloxavir at \u0026ge;\u0026thinsp;10 mg/kg protected mice from lethal A(H5N1) infection and prevented virus replication in the lungs and brain.\u003c/p\u003e\n\u003ch3\u003eFavipiravir efficacy\u003c/h3\u003e\n\u003cp\u003eFavipiravir administered at +\u0026thinsp;24 hpi improved the survival of mice challenged with A/scaup/GA/22 in a dose-dependent manner. Treatment with favipiravir at 20 mg/kg/day resulted in 0% survival, whereas 100 and 300 mg/kg/day resulted in 40% and 60% survival, respectively, and prolonged MSTs of 12.6 and 15.2 days, respectively (Fig.\u0026nbsp;3a,b). The changes in body weight and clinical scores for mice receiving 100 mg/kg/day and those receiving 300 mg/kg/day did not differ significantly (Fig.\u0026nbsp;3c,d). Favipiravir treatment at 100 and 300 mg/kg/day doses reduced viral titers in the lungs and brains of mice (Fig.\u0026nbsp;3e,f). The extent of pulmonary lesions and antigen distribution were markedly reduced in the higher-dose group (Fig.\u0026nbsp;3g,h). Overall, favipiravir treatment at \u0026ge;\u0026thinsp;100 mg/kg/day provided partial protection against lethal A(H5N1) infection.\u003c/p\u003e\n\u003ch3\u003eAmantadine efficacy\u003c/h3\u003e\n\u003cp\u003eDelayed amantadine treatment initiated at +\u0026thinsp;24 hpi with 15, 30, or 300 mg/kg/day did not improve the survival, MST, weight loss, or clinical score of mice challenged with A/scaup/GA/22 (Fig.\u0026nbsp;4a-d). This regimen did not reduce the lung and brain viral titers relative to those in untreated control mice (Fig.\u0026nbsp;4e,f). Importantly, mock-infected mice treated with 300 mg/kg/day of amantadine exhibited significant weight loss and elevated clinical scores indicating possible drug toxicity (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Overall, amantadine in all tested treatment regimens failed to protect mice lethally challenged with A(H5N1) clade 2.3.4.4b viruses.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEmergence of drug-resistant variants and host adaptation\u003c/h2\u003e\u003cp\u003eWe conducted deep sequencing of 14 paired lung and brain samples from mice that were treated with the highest dosage of each antiviral and of 10 samples from control animals (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We focused on substitutions in the proteins targeted by each drug, e.g., NA, M2, PA, or PB1, with significance being given to substitutions present in \u0026ge;\u0026thinsp;5% of the viral population and/or that were previously associated with clinical reports of treatment-emergent reduced susceptibility variants. No such substitutions in the NA and PB1 proteins were identified in oseltamivir-treated and favipiravir-treated mice, respectively. One animal treated with amantadine exhibited M2-S31N, the most frequently identified marker of amantadine resistance in seasonal influenza A viruses, as a minor variant (19%). Five previously unreported amino acid substitutions in the N-terminal domain (M2-K12R, M2-N18K, M2-S20N) or C-terminal domain (M2-V51I, M2-G61R) of the M2 ion channel were detected in more than 95% of the viral population in the brain of four amantadine-treated mice. Phenotypic testing of variants carrying these five M2 substitutions did not show reduced susceptibility to amantadine (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PA-I38L, associated with reduced baloxavir susceptibility in vitro\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, was predominant (present in \u0026ge;\u0026thinsp;95% of the virus population) in both lung and brain tissues of a single baloxavir-treated mouse. Overall, the NA, PA, and PB1 genetic markers of reduced antiviral susceptibility, including the clinically relevant markers NA-H275Y, NA-E199A/D/G (N1 numbering) (for NAIs), PA-I38T (for baloxavir), and PB1-K229R (for favipiravir) were not identified in drug-treated mice.\u003c/p\u003e\u003cp\u003eA portion of variants isolated from both drug-treated and untreated mice carried markers of mammalian host adaptation. PB2-E627K, which enhances viral replication and transmission in mammals\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, was identified as a minor variant (in 9.6%\u0026ndash;32.5% of viruses) in lungs of four of six animals treated with oseltamivir and favipiravir. One animal in the control group had this substitution predominating in both lungs (88%) and brain (\u0026ge;\u0026thinsp;95%) samples. PB2-D701N was found in lung and brain tissues of mice in different treatment groups and in vehicle controls. This substitution within the nuclear localization signal domain of the PB2 protein increases pathogenicity in mice\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and humans\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e by promoting nuclear import of viral influenza ribonucleoproteins\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. One animal exhibited minor variants with dual PB2-E627K and PB2-D701N substitutions, which enhance virulence and transmission in mice\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe 493\u0026ndash;512 region of the PA C-terminal domain is involved in structural interactions with the host transcription factor hCLE, which modulates nuclear RNA metabolism and enhances viral polymerase activity\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this region, we identified numerous instances of PA-C489S, which was particularly predominant in brain tissues (being present in \u0026ge;\u0026thinsp;95% of viruses). Additionally, PB2-L207I localized within the NP- and PB1-binding sites of the PB2 protein and was identified as a minor variant in lung tissue of oseltamivir- and amantadine-treated animals. It was also predominant in both tissues of two of five control mice. These previously unreported substitutions were considered because of their appearance in multiple animals and treatment groups.\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eHPAI A(H5N1) clade 2.3.4.4b viruses are characterized by elevated pathogenicity in avian and mammalian species, with potential for virus spread beyond the respiratory tract\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Therefore, there is significant concern regarding whether such viruses will cause severe disease in exposed humans and whether the available antivirals can control such infections. In this study, we comprehensively evaluated the potential therapeutic efficacy of four classes of antivirals that target different influenza virus proteins, including those targeting two polymerases and proteins mediating both entry and egress of A(H5N1) clade 2.3.4.4b viruses in mice. Our challenge viruses were the avian-origin A/scaup/GA/22 and A/hawk/NC/22 viruses, which exhibit 100% lethality with viral dissemination in mice\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Because the pathogenicity of these viruses is more severe than that of seasonal influenza viruses, we not only evaluated mouse treatment doses approximating human equivalents but also doses 5- to 10-fold greater\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Clinical guidance and pharmacokinetic studies were also taken into consideration when selecting the doses\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. According to the CDC Interim Guidance on treating novel influenza infections associated with severe human disease (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/bird-flu/hcp/clinicians-evaluating-patients/interim-guidance-treatment-humans.html\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/bird-flu/hcp/clinicians-evaluating-patients/interim-guidance-treatment-humans.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, July 3, 2025), treatment longer than 5 days is recommended for hospitalized patients with severe and prolonged illness. A 2-fold greater dose of oseltamivir has been proposed for treating influenza in immunocompromised patients and/or patients exhibiting progressive disease despite early administration of standard-dose medication\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNAIs are currently the first-line countermeasures against pandemic influenza\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, with more than 80% of the U.S. Strategic National Stockpile consisting of orally administered oseltamivir\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Oseltamivir IC\u003csub\u003e50\u003c/sub\u003es of HPAI A(H5N1) clade 2.3.4.4b viruses, including a recent human A/Texas/37/2024 isolate, were in the low-nanomolar range, \u0026lt;\u0026thinsp;10-fold IC\u003csub\u003e50\u003c/sub\u003e change vs. subtype-matched reference virus values, which aligns with previously reported data and meets the WHO criteria for drug susceptibility\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Oseltamivir at 20 mg/kg/day did not protect mice against HPAI A(H5N1) clade 2.3.4.4b virus infection, despite this dose reportedly producing a plasma concentration comparable to that of the recommended human oral dose of 75 mg BID\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Increasing the oseltamivir dose 5- to 10-fold provided 20%\u0026ndash;60% greater survival. Importantly, oseltamivir did not significantly reduce virus titers in lungs or brains. This may in part be due to the mechanism of NAIs, which primarily target the final step in the viral cycle (budding and egress of the progeny virions), meaning that one or more cycles have been completed in the infected cell(s), along with possible induction of host-cell inflammatory and antiviral responses\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In a recent study, oseltamivir (40 mg/kg/day) and zanamivir (8 mg/kg/day) administered orally BID for 5 days were unable to prevent lethality in mice infected with bovine-origin A(H5N1) 2.3.4.4b virus\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, a consistent regimen of baloxavir marboxil (50 mg/kg/day) protected 100% of mice against lethal bovine A(H5N1) infection\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, although the drug efficacy was significantly reduced when treatment was delayed until 48\u0026ndash;72 hpi\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAdditionally, consistent with previously published data on earlier A(H5N1) viruses\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we found no correlation between the survival of mice treated with clinically relevant doses of oseltamivir and \u003cem\u003ein vitro\u003c/em\u003e susceptibility. Therefore, for oseltamivir, and possible for other NAI drugs, the \u003cem\u003ein vitro\u003c/em\u003e phenotypic susceptibility of A(H5N1) clade 2.3.4.4b viruses may not reliably predict \u003cem\u003ein vivo\u003c/em\u003e treatment outcomes. Clinical recommendations for treating sporadic human A(H5N1) infections are primarily based on limited case series of patients and on extrapolation from seasonal influenza practicies\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies may be considered to include risk assessment practices and pharmacological management of emerging influenza viruses to confirm antiviral efficacy in animal models.\u003c/p\u003e\u003cp\u003eBaloxavir was the most successful of the four drugs studied with regard to its capacity to control virus replication in the lungs and brain. The 10 mg/kg dose resulted in 80% survival and significantly reduced virus titer in lungs and brains. This exceeds the 40% survival rate in mice treated with baloxavir marboxil at 50 mg/kg/day BID for 5 days, also starting at +\u0026thinsp;24 hpi\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This may be attributed to differences in the pharmacokinetics of the prodrug and its active metabolite. For critical cases of A(H5N6) infection in humans, baloxavir decreased the viral load and respiratory and serum cytokines, even when treatment was delayed for 3\u0026ndash;4 days. However, these data are complicated by the use of adjunct treatments, including NAIs, in these patients, as well as an overall lack of clinical reports of baloxavir use in humans infected with A(H5Nx) viruses\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Therefore, preclinical studies remain important potential indicators of drug usage in severe disease cases. Baloxavir targets the PA endonuclease activity, which cleaves capped host pre-mRNAs that can then be used by the virus for its own transcription. As this step occurs earlier in the viral replication cycle than the steps affected by NAIs, targeting it may provide faster arrest of the viral cycle and progeny virus propagation and could play a key role in controlling infections caused by HPAI A(H5N1) viruses, which are characterized by a highly efficient polymerase complex\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFavipiravir affects polymerase activity through a mechanism different from that of baloxavir\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Favipiravir targets the PB1 protein of the RNA polymerase complex\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, causing chain termination or lethal mutagenesis and inhibiting viral RNA synthesis slightly downstream of baloxavir but still upstream of NAI inhibition in the viral cycle. Therefore, drugs that act on these steps may provide enhanced benefits similar to those of CENI polymerase inhibitors. However, they currently have limited approval only in Japan because of teratogenicity reported in preclinical animal studies (Pharmaceuticals and Medical Devices Agency. Review report on Avigan, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.pmda.go.jp/files/000210319.pdf\u003c/span\u003e\u003cspan address=\"https://www.pmda.go.jp/files/000210319.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed August 25, 2025). Our data suggest that favipiravir provides some survival benefits and limits viral replication in lungs and brain, but it is less effective than baloxavir in terms of reducing morbidity, mortality, and some measures of viral presence in tissues. A(H1N1)pdm09 viruses with the PA-L666F substitution have been isolated from patients after favipiravir administration,\u003csup\u003e50\u003c/sup\u003e although the effect of the substitution on favipiravir susceptibility has not been reported.\u003c/p\u003e\u003cp\u003eOne virus isolated from a control animal has acquired PB2-E627K, a known virulence-associated marker of mammalian host adaptation\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough seasonal human influenza viruses are largely resistant to amantadine, we included this drug in our study as A(H5N1) viruses recently isolated from humans and birds were susceptible to M2 ion-channel blockers \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Importantly, adamantanes are the only class of influenza antivirals that exhibit saturable transport across the blood\u0026ndash;brain barrier\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, potentially enabling them to control A(H5N1) virus spread within the brain. The frequency of the M2-S31N variant that predominates in most amantadine-resistant seasonal influenza viruses remains low among A(H5N1) strains at \u0026lt;\u0026thinsp;0.76% (200 of 26,470 strains) (GISAID, Jan 1, 2022 \u0026ndash; Aug 15, 2025). Amantadine at 30 mg/kg/day reportedly protected 60% of mice lethally challenged with amantadine-susceptible A/Vietnam/1203/2004 (H5N1) clade 1 virus\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, and another study showed amantadine pre-exposure prophylaxis to be 100% effective against lethal A(H5N1) infection\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In contrast, our study demonstrated the inefficiency of amantadine against currently circulating HPAI A(H5N1) clade 2.3.4.4b viruses, which was not due to the development of drug-resistant variants. Amantadine\u0026rsquo;s target M2 protein facilitates HA protein fusion of influenza virions with host endosomal membranes by modulating and lowering the pH of the endosome. The fusion pH for the H5 HA protein of bovine-origin influenza A(H5N1) clade 2.3.4.4b viruses is 5.9, outside the pH range associated with human-like receptor binding (pH 5.0\u0026ndash;5.5)\u003csup\u003e56\u003c/sup\u003e, although earlier studies found mutations within the fusion peptide pocket of H5 HA that changed the pH of activation\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The lower pH of fusion in contemporary A(H5N1) viruses may make them less dependent on M2 ion channel\u0026ndash;mediated endosomal pH acidification, which could make amantadine antiviral mechanisms less effective \u003cem\u003ein vivo\u003c/em\u003e. However, rapid dissemination of virus into multiple organs during early stages of infection may also limit therapeutic efficacy, particularly with late treatment initiation.\u003c/p\u003e\u003cp\u003eBecause of the high pathogenicity and neuroinvasiveness of HPAI A(H5N1) viruses\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, the ability of antivirals to affect early stages of virus replication is crucial to controlling infections by these viruses. Among the four direct-acting antivirals studied, the polymerase inhibitors are the most promising drugs. A previous study demonstrated that the PA and, to a lesser extent, PB2 subunits of the polymerase were responsible for the increased polymerase activity of HPAI A(H5N1) viruses\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Baloxavir targets one of these proteins and effectively limited viral titers early in infection/treatment in adolescent and adult outpatients with uncomplicated influenza\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Baloxavir was also associated with a significant decline in virus titers and viral RNA load when compared with oseltamivir in Phase III trials\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Patients who received baloxavir experienced significantly faster resolution of hypoxia when compared with oseltamivir recipients\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In a recent pediatric study in Japan, a single baloxavir dose shortened symptom duration, with early reduction in viral titer, when compared with a 5-day course of oseltamivir\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBaloxavir inhibition of the polymerase complex during early stages of infection may prevent the systemic virus spread that is responsible for multiorgan failure and lethality. By contrast, NAIs such as oseltamivir target the last stage of the viral cycle and may be less effective at limiting the initial rounds of viral replication. Our data showed that NAI efficacy was associated with therapy initiation time. Pre-exposure prophylaxis with oseltamivir, beginning at \u0026minus;\u0026thinsp;4 hpi and using a consistent regimen, improved the survival rate by 20% when compared with delayed treatment (initiated at +\u0026thinsp;24 hpi). Accelerated failure time modeling showed that a shorter initiation of treatment with oseltamivir reduced illness duration and symptom severity when compared with intervention at 48 h after fever onset\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Therapeutic benefits have been found in patients with acute influenza infection when oseltamivir treatment was initiated no later than 36\u0026thinsp;\u0026minus;\u0026thinsp;48 h after the onset of symptoms, indicating the importance of early administration\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn summary, our study demonstrates that among four classes of influenza antivirals with wide or limited approval, only the polymerase inhibitor baloxavir provides consistent and robust protection and reduction in viral titers after lethal challenge with A(H5N1) clade 2.3.4.4b in mice. The polymerase inhibitor favipiravir provided partial protection against both disease criteria. In contrast, oseltamivir afforded limited survival benefits and did not control viral titers. These results may in part be due to targeting the early steps of the viral replication cycle and may be of significance for A(H5N1) viruses that have increased polymerase activity. For amantadine and oseltamivir, we found little to no correlation between their antiviral efficacy in mice and susceptibility \u003cem\u003ein vitro\u003c/em\u003e, confirming previously published data\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. At present, we encourage prioritization of baloxavir or, potentially, baloxavir drug combinations as a potential frontline therapeutic approach for A(H5N1) clade 2.3.4.4b infections. We also suggest that more prognostic approaches are warranted to evaluate antivirals against emerging influenza viruses.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEthics statements and biosafety\u003c/h2\u003e\u003cp\u003eAll protocols and procedures were approved by the St. Jude Children\u0026rsquo;s Research Hospital Institutional Animal Care and Use Committee and complied with the policies of the National Institutes of Health and the Animal Welfare Act. The animal experiments were conducted in an Animal Biosafety Level 3\u0026thinsp;+\u0026thinsp;containment facility in accordance with the U.S. Federal Select Agent Program regulations (7 CFR Part 331, 9 CFR Part 121.3, 42 CFR Part 73.3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eViruses, cells, and compounds\u003c/h2\u003e\u003cp\u003eInfluenza A(H5N1) viruses were propagated in the allantoic cavities of 10-day-old embryonated chicken eggs at 35\u0026deg;C for up to 48 h. Influenza A(H1N1)pdm09 reference viruses were grown in Madin\u0026ndash;Darby canine kidney (MDCK) cells at 37\u0026deg;C for up to 72 h. Aliquots were stored at \u0026minus;\u0026thinsp;80\u0026deg;C before use. MDCK cells obtained from the American Type Culture Collection were grown in culture in Modified Eagle\u0026rsquo;s Medium (MEM) (Thermo Fisher) supplemented with 5% fetal bovine serum (HyClone), 1 mM L-glutamine, and 1\u0026times; penicillin/streptomycin/amphotericin B (Gibco). Oseltamivir carboxylate and phosphate, baloxavir acid, and favipiravir were purchased from MedChem Express, amantadine was purchased from Sigma-Aldrich. Oseltamivir carboxylate/phosphate and amantadine were resuspended in ultrapure sterile water; stock solutions for baloxavir acid and favipiravir were prepared in sterile DMSO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNAI susceptibility\u003c/h2\u003e\u003cp\u003ePhenotypic susceptibility to oseltamivir carboxylate was determined by a fluorescence-based assay measuring the ability of the NA protein to cleave 2\u0026prime;-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA) substrate\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The NA activity of tested influenza viruses was standardized to relative fluorescent units equivalent to 10 \u0026micro;M 4-methylumbelliferone sodium salt (4-MU) (Sigma-Aldrich), and the viruses were incubated with oseltamivir carboxylate (5 pM to 50 \u0026micro;M) at 37\u0026deg;C for 30 min. The fluorescence signal from the NA-cleaved fluorogenic MUNANA substrate was measured at excitation/emission wavelengths of 360/460 nm. Half-maximal inhibitory concentrations (IC\u003csub\u003e50s\u003c/sub\u003e) were estimated from dose\u0026ndash;response curves by using a sigmoidal, four-parameter logistic nonlinear regression equation (in GraphPad Prism v.10.1.2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCENI susceptibility\u003c/h2\u003e\u003cp\u003ePhenotypic susceptibility to baloxavir was determined by an NA-based replication inhibition assay in MDCK cells\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Virus inoculum was normalized to 1.9 nM/well of 4-MU and was incubated with baloxavir (6 pM to 111 nM) on MDCK cells at 37\u0026deg;C for 12 h without TPCK-treated trypsin. The NA activity of the cell-attached virus was measured as an indicator of PA activity, and baloxavir half-maximal effective concentrations (EC\u003csub\u003e50s\u003c/sub\u003e) were calculated as described above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eFavipiravir and amantadine susceptibility\u003c/h2\u003e\u003cp\u003ePhenotypic susceptibility to favipiravir and amantadine was determined by plaque-reduction assays. MDCK cells were inoculated with virus at a dose yielding 50\u0026ndash;100 plaque-forming units (PFUs). After incubation, cell monolayers were washed and overlaid with 0.45% immunodiffusion-grade agarose (MP Biomedical) in DMEM with 4% bovine serum albumin, 1 \u0026micro;g/mL TPCK-treated trypsin (Worthington Biochemical) (for non-A(H5N1) viruses), and 10-fold dilutions of favipiravir/amantadine (1 nM to 1 mM). For amantadine, the inoculum was pre-incubated with amantadine at corresponding concentrations at 37\u0026deg;C for 1 h. At 48\u0026ndash;72 hpi, cells were fixed with 10% formaldehyde and stained with 1% crystal violet, and the PFUs per well were enumerated. EC\u003csub\u003e50\u003c/sub\u003es were determined by using the log (inhibitor) versus response logistic nonlinear regression equation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAntiviral efficacy in mice\u003c/h2\u003e\u003cp\u003eFemale 6-week-old BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were lightly anesthetized with isoflurane and inoculated intranasally with 5 \u0026times; 50% mouse lethal doses (MLD\u003csub\u003e50\u003c/sub\u003es) of each virus in 50 \u0026micro;L of PBS. Starting at 24 hpi, mice (n\u0026thinsp;=\u0026thinsp;5/group) were treated by oral gavage (100 \u0026micro;L/mouse) BID (at 12 h intervals) for 5 days with 20, 100, or 200 mg/kg/day of oseltamivir; 20, 100, or 300 mg/kg/day of favipiravir; or 30 or 300 mg/kg/day of amantadine. Baloxavir acid was administered subcutaneously as a single dose at 0.1, 1, or 10 mg/kg (100 \u0026micro;L/mouse). Control animals (n\u0026thinsp;=\u0026thinsp;5/group) received 100 \u0026micro;L of vehicle (ORA-Plus\u003csup\u003e\u0026reg;\u003c/sup\u003e/water) orally BID for 5 days. For the prophylactic regimens, administration of oseltamivir or baloxavir was initiated at \u0026minus;\u0026thinsp;4 hpi. Mice were weighed daily and monitored for morbidity and mortality (death or loss of \u0026ge;\u0026thinsp;25% of their initial body weight) for 21 dpi (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). A daily clinical score for each group was based on the clinical signs of disease, such as lethargy, ruffled fur, hunched posture, respiratory distress (labored breathing), and neurologic complications. MST was calculated by the Kaplan\u0026ndash;Meier method\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. For tissue titration, mice (n\u0026thinsp;=\u0026thinsp;3/group) were euthanized at 3 and 6 dpi, and the brains and lungs were collected and homogenized with a TissueLyser system (Qiagen) in 1 mL of MEM. The homogenates were cleared by centrifugation at 3000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min and used to determine 50% tissue culture infectious doses (TCID\u003csub\u003e50\u003c/sub\u003es) by inoculating log10 dilutions onto MDCK cells (with incubation at 37\u0026deg;C for 3 days). TCID\u003csub\u003e50\u003c/sub\u003e values were determined by the method of Reed and Muench\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHistopathology and immunohistochemistry\u003c/h2\u003e\u003cp\u003eAt 3 and 6 dpi, mice (n\u0026thinsp;=\u0026thinsp;3/group) were euthanized, and the lungs and nasal turbinates were infused via the trachea with 10% neutral-buffered formalin (NBF) (Thermo Fisher). The lungs and brains were then collected and fixed by immersion in 10% NBF before being embedded, sectioned, and stained with hematoxylin and eosin (HE) or subjected to immunohistochemical labeling with a rabbit monoclonal antibody/to influenza A virus nucleoprotein (GTX636247, GeneTex, Irvine, CA). Slides were scanned and the percentage areas of lung parenchyma classified as normal versus virus-positive/inflamed were determined by quantitative morphometry using the HALO\u0026trade; MiniNet AI algorithm (IndicaLabs, Albuquerque, NM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eSequencing\u003c/h2\u003e\u003cp\u003eViral RNA was isolated from mouse lung and brain homogenates with an RNeasy Kit (Qiagen). PA, NA, M2, PB1 and PB2 gene segments were amplified from total RNA with universal or gene-specific primers and purified with a QIAquick PCR Purification Kit (Qiagen). Libraries were prepared with a Nextera XT DNA Library Prep Kit (Illumina) and sequenced with a MiSeq Reagent Kit v2 (300 cycles) on a MiSeq System (Illumina). Reads were quality trimmed and assembled with CLC Genomics Workbench v24 (Qiagen). Amino acid variants were called with a variant frequency cutoff of 5% for \u0026ge;\u0026thinsp;1000 reads. Consensus sequences were aligned using BioEdit v7.0.9 software\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Data analysis was based on NA and PA molecular markers associated with reduced or highly reduced inhibition by NAIs or the CENI as summarized by the World Health Organization Antiviral Working Group (Summary of NA amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors; (PA) amino acid substitutions analyzed for their effects on baloxavir susceptibility). PB1-K229R was monitored for resistance to favipiravir\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. M2 substitutions at residues 26, 27, 30, 31, and 34 were monitored for resistance to amantadine.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eSerology\u003c/h2\u003e\u003cp\u003ePost-infection mouse serum samples were obtained at 21 dpi, treated with a receptor-destroying enzyme (Denka), heat inactivated at 56\u0026deg;C for 1 h, and tested for the presence of anti-HA antibodies by performing HA inhibition assays with chicken red blood cells. The reciprocal of the last serum dilution that inhibited hemagglutination was recorded as the HI titer (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed by GraphPad Prism 10.1.2 software (La Jolla, CA), with individual significance being determined by unpaired \u003cem\u003et-\u003c/em\u003etests and/or one-way analysis of variance (ANOVA).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSusceptibility of A(H5N1) viruses to the NAI oseltamivir carboxylate, the CENI baloxavir, and the RNA-dependent RNA polymerase inhibitor favipiravir.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInfluenza A virus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOseltamivir [nM]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBaloxavir [nM]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eT-705 [\u0026micro;M]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA(H5N1) viruses\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/red-shouldered hawk/North Carolina/W22-121/2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/lesser scaup/Georgia/W22-145E/2022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/Texas/37/2024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e12.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/Vietnam/1203/2004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA(H1N1)pdm09 reference viruses\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/Denmark/528/2009 \u003cb\u003eNA-H275Y\u003c/b\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e137.33\u003c/b\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/Illinois/08/2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/Illinois/308/2018 \u003cb\u003ePA-I38T\u003c/b\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e34.53\u003c/b\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;16.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e The results are representative of two or three independent dose\u0026ndash;response curves\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard deviation (SD).\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e N1 numbering.\u003c/p\u003e\u003cp\u003eNA/PA substitutions that conferred reduced inhibition/highly reduced inhibition by oseltamivir/baloxavir (IC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e fold-change\u0026thinsp;\u0026ge;\u0026thinsp;10/\u0026gt;3) are indicated in bold.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in manuscript figures and text.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis project was funded by the U.S. NIAID, U.S. NIH, and U.S. DHHS under contract 75N93021C00016. This content is the responsibility of the authors and does not necessarily represent official views of the U.S. NIH.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions:\u003c/h2\u003e\n\u003cp\u003eKA, RJW and EAG conceptualized the research project. KA, JCJ, AK, PV acquired and/or analyzed the data. KA, JCJ and EAG wrote original drafts, and all authors reviewed and edited subsequent drafts. Funding was acquired by RJW.\u003c/p\u003e\n\u003ch2\u003eACKNOWLEDGMENTS:\u003c/h2\u003e\n\u003cp\u003eThe authors thank Keith A. Laycock, PhD, ELS, for scientific editing of the manuscript, David Carey, Heather Weinberg, and Chelsi Stultz for animal husbandry, the St. Jude Hartwell Center for Bioinformatics and Biotechnology for next-generation sequencing, Patrick Seiler, Jeri-Carol Crumpton for experimental assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNair H et al (2011) Global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. Lancet 378:1917\u0026ndash;1930\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSwayne DE et al (2024) Strategic challenges in the global control of high pathogenicity avian influenza. Rev Sci Tech Special Ed, 89\u0026ndash;102\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarg S et al (2025) Highly pathogenic avian influenza A(H5N1) virus infections in humans. N Engl J Med 392:843\u0026ndash;854\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu X, Subbarao, Cox NJ, Guo Y (1999) Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261:15\u0026ndash;19\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePardo-Roa C et al (2025) Cross-species and mammal-to-mammal transmission of clade 2.3.4.4b highly pathogenic avian influenza A/H5N1 with PB2 adaptations. Nat Commun 16:2232\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRolfes MA et al (2025) Human infections with highly pathogenic avian influenza A(H5N1) viruses in the United States from March 2024 to May 2025. 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National Academies\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWard P, Small I, Smith J, Suter P, Dutkowski R (2005) Oseltamivir (Tamiflu) and its potential for use in the event of an influenza pandemic. J Antimicrob Chemother 55(Suppl 1):i5\u0026ndash;i21\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoregian A, Mercorelli B, Nannetti G, Compagnin C, Pal\u0026ugrave; G (2014) Antiviral strategies against influenza virus: towards new therapeutic approaches. Cell Mol Life Sci 71:3659\u0026ndash;3683\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGu C et al (2024) A human isolate of bovine H5N1 is transmissible and lethal in animal models. Nature 636:711\u0026ndash;718\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKiso M, Uraki R, Yamayoshi S, Kawaoka Y (2025) Efficacy of baloxavir marboxil against bovine H5N1 virus in mice. 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J Exp Med 203:689\u0026ndash;697\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFang Q, Wang D (2020) Advanced researches on the inhibition of influenza virus by Favipiravir and Baloxavir. Biosaf Health 2:64\u0026ndash;70\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFuruta Y et al (2005) Mechanism of action of T-705 against influenza virus. Antimicrob Agents Chemother 49:981\u0026ndash;986\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakashita E et al (2016) Antiviral susceptibility of influenza viruses isolated from patients pre- and post-administration of favipiravir. Antiviral Res 132:170\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMin J-Y et al (2013) Mammalian adaptation in the PB2 gene of avian H5N1 influenza virus. J Virol 87:10884\u0026ndash;10888\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePascua PNQ et al (2025) Antiviral susceptibility of influenza A(H5N1) clade 2.3.2.1c and 2.3.4.4b viruses from humans, 2023\u0026ndash;2024. Emerg Infect Dis 31:751\u0026ndash;760\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpector R (1988) Transport of amantadine and rimantadine through the blood\u0026ndash;brain barrier. J Pharmacol Exp Ther 244:516\u0026ndash;519\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIlyushina NA, Hoffmann E, Salomon R, Webster RG, Govorkova EA (2007) Amantadine\u0026ndash;oseltamivir combination therapy for H5N1 influenza virus infection in mice. Antivir Ther 12:363\u0026ndash;370\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSmee DF, Hurst BL, Wong MH, Bailey KW, Morrey JD (2009) Effects of double combinations of amantadine, oseltamivir, and ribavirin on influenza A (H5N1) virus infections in cell culture and in mice. Antimicrob Agents Chemother 53:2120\u0026ndash;2128\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang J et al (2025) The haemagglutinin gene of bovine-origin H5N1 influenza viruses currently retains receptor-binding and pH-fusion characteristics of avian host phenotype. Emerg Microbes Infect 14:2451052\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReed ML et al (2009) Amino acid residues in the fusion peptide pocket regulate the pH of activation of the H5N1 influenza virus hemagglutinin protein. J Virol 83:3568\u0026ndash;3580\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBauer L, Benavides FFW, Kroeze V, de Wit EJB, E., van Riel D (2023) The neuropathogenesis of highly pathogenic avian influenza H5Nx viruses in mammalian species including humans. Trends Neurosci 46:953\u0026ndash;970\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLeung BW, Chen H, Brownlee GG (2010) Correlation between polymerase activity and pathogenicity in two duck H5N1 influenza viruses suggests that the polymerase contributes to pathogenicity. Virology 401:96\u0026ndash;106\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIson MG et al (2020) Early treatment with baloxavir marboxil in high-risk adolescent and adult outpatients with uncomplicated influenza (CAPSTONE-2): a randomised, placebo-controlled, phase 3 trial. Lancet Infect Dis 20:1204\u0026ndash;1214\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKuo Y-C, Lai C-C, Wang Y-H, Chen C-H, Wang C-Y (2021) Clinical efficacy and safety of baloxavir marboxil in the treatment of influenza: a systematic review and meta-analysis of randomized controlled trials. J Microbiol Immunol Infect 54:865\u0026ndash;875\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShah S et al (2020) Clinical outcomes of baloxavir versus oseltamivir in patients hospitalized with influenza A. J Antimicrob Chemother 75:3015\u0026ndash;3022\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshiguro N et al (2025) Clinical and virologic outcomes of baloxavir compared with oseltamivir in pediatric patients with influenza in Japan. Infect Dis Ther 14:833\u0026ndash;846\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAoki FY et al (2003) Early administration of oral oseltamivir increases the benefits of influenza treatment. 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J Am Stat Assoc 53:457\u0026ndash;481\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eReed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493\u0026ndash;497\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: \u003cem\u003eNucleic Acids Symposium Series No. 41.\u003c/em\u003eOxford University Press, pp. 95\u0026ndash;98\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoldhill DH et al (2018), \u003cem\u003eThe mechanism of resistance to favipiravir in influenza. Proc. Natl. Acad. Sci. U. S. A. 115, 11613\u0026ndash;11618\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7768971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7768971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntercontinental spread of highly pathogenic avian influenza A(H5N1) viruses poses significant pandemic risks and necessitates strong protective countermeasures. We evaluated the therapeutic efficacy of the neuraminidase inhibitor oseltamivir, the polymerase inhibitors baloxavir and favipiravir, and an ion-channel blocker amantadine, against severe influenza A(H5N1) infection in mice. Oseltamivir (\u0026ge;\u0026thinsp;100 mg/kg/day for 5 days) provided limited survival benefits and failed to prevent viral neuroinvasion. Baloxavir (\u0026ge;\u0026thinsp;10 mg/kg, 1 dose) fully protected mice, significantly reduced virus respiratory replication, and prevented neuroinvasion. Favipiravir (\u0026ge;\u0026thinsp;100 mg/kg/day for 5 days) provided partial protection, with viral titers being reduced in lungs and brain. Amantadine provided no benefits. Although all drugs inhibited A(H5N1) viruses \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003ein vivo\u003c/em\u003e correlations did not extend beyond baloxavir. Our results indicate that baloxavir is the most reliable treatment to address both respiratory replication and systemic spread of contemporary A(H5N1) viruses in mice and should be considered in pandemic planning.\u003c/p\u003e","manuscriptTitle":"Baloxavir outperforms oseltamivir, favipiravir, and amantadine in treating lethal influenza A(H5N1) HA clade 2.3.4.4b infection in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 14:53:58","doi":"10.21203/rs.3.rs-7768971/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ba3a3c1-ac71-417a-bd3b-549e3d312d29","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56053063,"name":"Biological sciences/Microbiology/Virology/Influenza virus"},{"id":56053064,"name":"Biological sciences/Microbiology/Virology/Antivirals"}],"tags":[],"updatedAt":"2026-03-28T07:13:26+00:00","versionOfRecord":{"articleIdentity":"rs-7768971","link":"https://doi.org/10.1038/s41467-026-69721-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-19 05:00:00","publishedOnDateReadable":"February 19th, 2026"},"versionCreatedAt":"2025-10-20 14:53:58","video":"","vorDoi":"10.1038/s41467-026-69721-5","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69721-5","workflowStages":[]},"version":"v1","identity":"rs-7768971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7768971","identity":"rs-7768971","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}