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

Domain: chemistry
Published: 2026-02-19
Editorial outcome: major_revision

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) virus infection in female BALB/c mice. Baloxavir (≥10 mg/kg, 1 dose) fully protected mice from death, significantly reduced virus respiratory replication, and prevented neuroinvasion. Oseltamivir (≥100 mg/kg/day for 5 days) provided limited survival benefits, reduced lung titers but failed to prevent viral neuroinvasion. Favipiravir (≥100 mg/kg/day for 5 days) provided partial protection, although did not reduce viral titers 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.

Introduction
Influenza viruses are significant human pathogens that cause respiratory disease and annual epidemics1. However, they are also infecting wild birds, domestic poultry, and other peridomesticated species2. This provides opportunities for zoonotic transmission to humans, with some influenza subtypes being capable of causing severe disease and death3. 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 19964. 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 humans5. 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 low6. 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 Mexico7,8 (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 models9.

Despite efforts to develop novel clade-specific vaccine candidates10,11, vaccines against circulating clade 2.3.3.4b viruses are not available in large quantities12. 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 isolates13 and those from virus-contaminated raw [unpasteurized] milk14) to U.S. Food and Drug Administration–approved antivirals in vitro15. 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 viral 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 CENIs16. 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 infections17, 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 humans18.

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 mutagenesis19. It is approved for use in Japan but with significant restrictions20. The adamantane class of drugs, including amantadine (Gocovri®) target the viral matrix 2 (M2) protein to prevent viral entry, but their use to treat seasonal influenza has been discontinued because of widespread resistance21,22. 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 viruses9 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 (IC50/EC50) were <10-fold and <3-fold greater for the NAI and CENI, respectively, compared to median IC50/EC50 values of circulating North American A(H5N1) clade 2.3.4.4b viruses16. 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 EC50 values were in the micromolar range, which is comparable to those for pandemic A(H1N1) pdm09 influenza viruses (Table 1) and to previously reported data23. Amantadine EC50 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) (Figs. 1–4a, c, S1a, b, S2, and S3a, c). The average morbidity (clinical scores) peaked progressively at 6 dpi (Figs. 1–4d and S2, S3d). More than 60% of control animals (13 of 20) displayed neurologic signs, including hind-limb paresis, ataxia, and tremors. The mean survival times (MSTs) of control mice upon infection with both viruses were ≤5 days (Figs. 1–4b and S2, S3b).

A/scaup/GA/22 titers in lungs of untreated mice were 106–107 TCID50/mL at 3 and 6 dpi, with brain titers rising from 102 to 107 TCID50/mL between 3 and 6 dpi (Figs. 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. S1c). 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. S1d). Perivascular inflammatory cell infiltration, 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. A substantial portion of variants isolated from A/scaup/GA/22-infected mice in both treated and control groups also carried markers of mammalian host adaptation, including PB2-E627K and PB2-D701N (Table S1).

Oseltamivir efficacy
We examined the benefits of pre-exposure prophylaxis and 24h–delayed treatment with the NAI oseltamivir in mice. Prophylactic oseltamivir initiated at −4h 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. S2a–d). Oseltamivir administered +24hpi 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 at ≥20 mg/kg/day reduced virus titers in the lung by about 2 log10 TCID50/mL by 6 dpi but not in the brain (Figs. 1e, f and S2e, 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 −4hpi 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. S3a–f). Delayed baloxavir treatment (initiated at +24hpi) 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). Baloxavir at ≥0.1 mg/kg significantly reduced virus replication in lungs and brain, with viral titers to be below the limit of detection upon administration of 25 mg/kg dose (Fig. 2e, f). Lungs from mice treated with 0.1 mg/kg were characterized by extensive central pneumonia, with 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 +24hpi 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 and in 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 ≤300 mg/kg/day did not reduce viral titers in the lungs and brains of mice (Fig. 3e, f), whereas the extent of pulmonary lesions and antigen distribution was 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 +24hpi 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 potential reduced susceptibility substitutions in the NA, M2, PA, or PB1 proteins targeted by oseltamivir, amantadine, baloxavir and favipiravir, respectively. The 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 vitro24, 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 mammals25, was identified as a minor variant (in 9.6%–32.5% of viruses) in the 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 the 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 mice26 and humans27 by promoting nuclear import of viral influenza ribonucleoproteins28. One animal exhibited minor variants with dual PB2-E627K and PB2-D701N substitutions, which enhance virulence and transmission in mice29.

The 493–512 region of the PA C-terminal domain is involved in structural interactions with the human CLE/C14orf166 protein, the host transcription factor that modulates nuclear RNA metabolism and enhances viral polymerase activity30. 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 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.

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 (protocol numbers 003 and 428) 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). Mice were housed in HEPA-filtered negative-pressure vented isolation containers with 12h dark/light cycle, +25°C temperature and 45% humidity.

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 48h. Influenza A(H1N1)pdm09 reference viruses were grown in Madin–Darby canine kidney (MDCK) cells (ATCC, CCL-34) at 37°C for up to 72h. Aliquots were stored at −80°C before use. GenBank accession numbers of gene segments for viruses used in this study are provided in Table S2. 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) substrate62. The NA activity of tested influenza viruses was standardized to relative fluorescent units equivalent to 10µM 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 360/460nm. Half-maximal inhibitory concentrations (IC50) 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 cells63. Virus inoculum was normalized to 1.9nM/well of 4-MU and was incubated with baloxavir (6 pM to 111 nM) on MDCK cells at 37°C for 12h 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 (EC50) 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, 1μg/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 1h. At 48-72 hpi, cells were fixed with 10% formaldehyde and stained with 1% crystal violet, and the PFUs per well were enumerated. EC50 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 (MLD50) in 50 µL of PBS of A/scaup/GA/22 (4.0 log10 TCID50/mouse) and A/hawk/NC/22 (4.1 log10 TCID50/mouse) virus. Starting at 24 hpi, mice (n = 5/group) were treated by oral gavage (100 µL/mouse) BID (at 12h 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 (based on beneficial pharmacokinetics properties of baloxavir acid under subcutaneous administration)35 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 method64. 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 Tissue Lyser 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 (TCID50s) by inoculating log10 dilutions onto MDCK cells (with incubation at 37°C for 3 days). TCID50 values were determined by the method of Reed and Muench65.

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 (Indica Labs, Albuquerque, NM).

Sequencing
Viral RNA was isolated from mouse lung and brain homogenates with a RNeasy Kit (Qiagen). PA, NA, M2, PB1, and PB2 gene segments were amplified from total RNA with universal primers (Table S3) 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 an next-generation sequencing (NGS) MiSeq System (Illumina) for NGS. 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 software66. 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 favipiravir67. 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 1h, 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 or one-/two-way analysis of variance (ANOVA). Replicates, group comparisons, and P values are listed in each figure legend.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall, this manuscript is extremely well written and the conclusions are supported by the data. I do not have any major concerns about this manuscript. A few minor concerns are listed below.\n1. In the discussion section (lines 318-320), the authors indicate that the lower pH of fusion in contemporary A(H5N1) viruses may reduce the dependence on M2 ion channel-mediated endosomal pH acidification. In line 315, the authors indicate that the pH of bovine-origin H5N1 influenza is 5.9. This supports the authors claim but is a higher pH rather than a lower pH unless I have misunderstood in some way.\n2. In the methods section (lines 411-413), the dose of virus used for infection is listed as MLD50s in 50 µL of PBS. Can that information be correlated to PFUs/mL or CCID50/mL per mouse?\n3. For the figure legends, the statistical analyses are not listed for each specific figure. The Statistical analysis section indicates that either unpaired t-tests and/or one-way ANOVAs were completed. I assume that the correct analyses were done but it would be ideal to have that information confirmed in the figure legends.", "reviewer_2": "This manuscript contains important results. However, some revisions are needed for publication. Especially Discussion looks redundant. It should be stated just on the basis of the present results. This reviewer would like to read the manuscript revised according to the following comments.\nline 26: influenza infection should be influenza virus infection. 62: incomplete sentence, put \")\".\n95: What is subtype-matched reference? 96: normal drug susceptibility of the two avian H5N1 viruses according to ---. 116-119: should be clearer expression. 126: perivascular inflammatory cell \"infiltration\", 188: What do amino acid substitutions mean? 215: full spell of CLE. 218: Last \"and\" should be deleted. It should be stated the reason why Baloxavir was administered subcutaneously.\nDiscussion: redundant, should be much more exactly stated just on the basis of the present results.\nWaiting for the manuscript clearly revised.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We greatly appreciate the thoughtful comments and suggestions of the editor and reviewers and have incorporated changes and modifications throughout the text of the revised manuscript. We modified the Discussion section to clarify reviewers’ comments. Please see below our point-by-point responses to reviewer’s comments.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nOverall, this manuscript is extremely well written, and the conclusions are supported by the data.\nI do not have any major concerns about this manuscript. A few minor concerns are listed below.\nCOMMENT #1. In the discussion section (lines 318-320), the authors indicate that the lower pH\nof fusion in contemporary A(H5N1) viruses may reduce the dependence on M2 ion channel-\nmediated endosomal pH acidification. In line 315, the authors indicate that the pH of bovine-\norigin H5N1 influenza is 5.9. This supports the author’s claim but is a higher pH rather than a\nlower pH unless I have misunderstood in some way.\nRESPONSE: We appreciate the reviewer’s comment and modified the text of the Discussion\nsection accordingly. For clarity of data description, we deleted the sentences about the fusion pH\nfor the H5 HA proteins of bovine-origin influenza A(H5N1) viruses. (lines 313-323 in the\noriginal manuscript).\nCOMMENT #2. In the methods section (lines 411-413), the dose of virus used for infection is\nlisted as MLD50s in 50 µL of PBS. Can that information be correlated to PFUs/mL or\nCCID50/mL per mouse?\nRESPONSE: We modified the text of the Method section according to reviewer’s comment and\nprovided the dose of influenza viruses used for challenge mice expressed as log TCID /mouse.\n10 50\nIt reads: “Female 6-week-old BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were lightly\nanesthetized with isoflurane and inoculated intranasally with 5 × 50% mouse lethal doses\n(MLD s) in 50 µL of PBS of A/scaup/GA/22 (4.0 log TCID /mouse) and A/hawk/NC/22 (4.1\n50 10 50\nlog TCID /mouse) virus”. (lines 411-414)\n10 50\nCOMMENT #3. For the figure legends, the statistical analyses are not listed for each specific\nfigure. The Statistical analysis section indicates that either unpaired t-tests and/or one-way\nANOVAs were completed. I assume that the correct analyses were done but it would be ideal to\nhave that information confirmed in the figure legends.\nRESPONSE: We modified the figure legends to Figures 1-4 accordingly and indicated the\nstatistical analysis used.\nThis manuscript contains important results. However, some revisions are needed for publication.\nCOMMENT #1. Discussion looks redundant. It should be stated just on the basis of the present\nresults.\nRESPONSE: We appreciate the reviewer’s suggestion. We have shortened the Discussion\nsection of the manuscript and removed discussions about the influence of fusion pH in\ncontemporary influenza A(H5N1) viruses on amantadine susceptibility (lines 313-323 in the\noriginal manuscript) and sentences about the effect of PA-L666F on favipiravir susceptibility\n(lines 301-306 in the original manuscript).\nCOMMENT #2. Line 26: influenza infection should be influenza virus infection.\nRESPONSE: The sentence has been changed accordingly. (line 28)\nCOMMENT #3. Line 62: incomplete sentence, put \")\".\nRESPONSE: The sentence has been changed accordingly. (line 65)\nCOMMENT #4. Line 95: What is subtype-matched reference?\nRESPONSE: The sentence has been changed accordingly. It reads: “The resulting 50%\ninhibitory/effective concentrations (IC /EC ) were <10-fold and <3-fold greater for the NAI\n50 50\nand CENI, respectively, compared to median IC /EC values of circulating North American\n50 50\nA(H5N1) clade 2.3.4.4b viruses (Andreev et al. Antiviral susceptibility of highly pathogenic\navian influenza A(H5N1) viruses circulating globally in 2022-2023. J Infect Dis 229(6): 1830-\n1835, 2024).” (lines 97-100)\nCOMMENT #5. Line 96: normal drug susceptibility of the two avian H5N1 viruses according\nto ---.\nRESPONSE: Normal drug susceptibility of the two avian A(H5N1) viruses was defined based\non “current WHO Antiviral Working Group (AVWG) criteria (World Health Organization\nAVWG Proposed NI Susceptibility Criteria for Surveillance and Reporting).” The source link is\nprovided as well. (lines 100-102)\nCOMMENT #6. Lines 116-119: should be clearer expression.\nRESPONSE: The sentences have been changed accordingly. It reads: “The average morbidity\n(clinical scores) peaked progressively at 6 dpi (Fig. 1-4d and Fig. S2-S3d). More than 60% of\ncontrol animals (13 of 20) displayed neurologic signs, including hind-limb paresis, ataxia, and\ntremors. The mean survival times (MSTs) of control mice upon infection with both viruses were\n≤ 5 days (Fig. 1-4b and Fig. S2-S3b).” (lines 119-123)\nCOMMENT #7. Line 126: perivascular inflammatory cell \"infiltration\".\nRESPONSE: The sentence has been changed accordingly. (line 130)\nCOMMENT #8. 188: What do amino acid substitutions mean?\npotential reduced susceptibility substitutions in the NA, M2, PA, or PB1 proteins targeted by\noseltamivir, amantadine, baloxavir and favipiravir, respectively”. (lines 194-196)\nCOMMENT #9. Line 215: full spell of CLE.\nRESPONSE: The influenza A virus polymerase associates with a number of cellular\ntranscription-related factors, including RNA polymerase II. We referred to previously described\ninteraction of influenza virus polymerase subunit PA with human CLE/C14orf166 protein\n(hCLE), a positive modulator of this cellular RNA polymerase (Rodriguez et al. Cellular human\nCLE/C14orf166 protein interacts with influenza virus polymerase and is required for viral\nreplication. J Virol. 85(22):12062-12066, 2011). The revised text now reads: “The 493–512\nregion of the PA C-terminal domain is involved in structural interactions with human\nCLE/C14orf166 protein, the host transcription factor that modulates nuclear RNA metabolism\nand enhances viral polymerase activity.” (lines 222 - 224)\nCOMMENT #10. Line 218: Last \"and\" should be deleted.\nRESPONSE: The sentence has been changed accordingly (line 227)\nCOMMENT #11. It should be stated the reason why Baloxavir was administered\nsubcutaneously.\nRESPONSE: Per reviewer’s comment, we stated the reason for subcutaneous administration of\nbaloxavir acid in a mouse model. It reads: Baloxavir acid was administered subcutaneously\n(based on beneficial pharmacokinetics properties of baloxavir acid under subcutaneous\nadministration) (Ando et al. Pharmacokinetic and pharmacodynamic analysis of baloxavir\nmarboxil, a novel cap-dependent endonuclease inhibitor, in a murine model of influenza virus\ninfection. J Antimicrob Chemother. 76(1):189-198, 2021) as a single dose at 0.1, 1, or 10 mg/kg\n(100 µL/mouse). (lines 417- 419)\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nRESPONSE: We did not receive any comments from Reviewer #3, however we are fully\nsupportive of this Nature Communications initiative.\n3"}]

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