Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus continues to cause animal outbreaks and sporadic zoonotic infections. In a mouse model of lethal influenza disease, we compared oseltamivir, baloxavir, and molnupiravir monotherapies with 2-drug combinations. Baloxavir-based combinations improved survival, reduced lung viral loads, and prevented extrapulmonary dissemination, supporting H5N1 preparedness strategies.

Recently, H9N2 avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Therefore, investigating the impact of its neuraminidase mutations on susceptibility to neuraminidase inhibitors (NAIs) is of great importance for clinically preventing potential cross-species transmission of H9N2. In this study, our results revealed that NAI resistance-related mutations of most H9N2 viruses isolated from China during 1998 to 2024 mainly occurred at positions N70S, E119A/D/G/V, Q133K, Q136A, D151E, Y155H, I222V, R224K, E276D, R292K, N294S, and R371K. Among them, the combined N70S and Y155H mutations are common characteristics of H9N2 viruses circulating in recent years. In vivo experiments demonstrated that these H9N2 viruses harboring the combined N70S and Y155H mutations exhibit high pathogenicity to BALB/c mice without prior adaption. In vitro neuraminidase inhibition assays confirmed that these H9N2 viruses bearing the N70S/Y155H mutations exhibited greater reduced inhibition than their counterparts bearing the N70S or Y155H single mutation to oseltamivir and zanamivir, but remained highly sensitive to peramivir. Notably, the in vivo protective effect of NAIs against H9N2 virus with combined N70S and Y155H mutations is limited, possibly due to the restricted ability of these drugs to ameliorate the excessive inflammatory responses. Therefore, there is an urgency to strengthen research on epidemiological surveillance and prevention strategies for avian influenza H9N2 harboring these mutations.

Roseofungin, a polyene macrolide antibiotic produced by Streptomyces roseoflavus, was evaluated for antiviral activity against Influenza A virus using complementary in vitro, in ovo, and preliminary in vivo toxicity models, supported by molecular docking and molecular dynamics simulations. The compound exhibited antiviral activity against multiple influenza A virus subtypes, including an oseltamivir-resistant H1N1 strain, with EC50 values ranging from 2.0 to 2.5 μg/mL and selectivity indices of 20-25. Functional assays demonstrated that roseofungin interfered with early stages of the viral life cycle by reducing viral adsorption and partially inhibiting neuraminidase activity. In virucidal assays, treatment with roseofungin resulted in a 1.5-2.5 log10 reduction in infectious viral titers. Acute toxicity studies in mice indicated low toxicity under the experimental conditions (LD50 > 25 mg/kg). Molecular docking predicted favorable interactions of roseofungin with multiple influenza A virus proteins. Subsequent molecular dynamics simulations of the hemagglutinin-roseofungin complex maintained the stability of the predicted binding mode across three independent 50 ns simulations (150 ns cumulative). Collectively, these findings demonstrate that roseofungin possesses broad-spectrum antiviral activity against genetically diverse Influenza A virus strains and represents a promising lead compound for the development of novel membrane-active antiviral agents. Further studies are warranted to elucidate its molecular mechanism of action and to evaluate its therapeutic potential in vivo.

Background/Objectives: Oseltamivir is a widely used antiviral agent indicated for the treatment of influenza and plays a central role in pandemic preparedness strategies. Although an extemporaneously prepared 15 mg/mL oral solution is recognized in formularies, preparing large volumes during a pandemic requires extended storage options. However, no stability studies are currently available for an oseltamivir 15 mg/mL oral solution stored under freezing and subsequent refrigerated conditions for the duration of a standard treatment. The aim of this study was to evaluate the physicochemical and microbiological stability of a 15 mg/mL oseltamivir oral solution prepared from the active pharmaceutical ingredient (API) and packaged in amber glass containers. Methods: The oral solution was formulated using oseltamivir phosphate API, sodium benzoate as a preservative, and purified water and then packaged in 125 mL Type II amber glass bottles, allowing for thermal expansion. The samples were stored at -20 ± 2 °C for up to 75 days, followed by refrigerated storage (5 ± 3 °C) after thawing for up to 10 days. Chemical stability was assessed using a validated HPLC method in accordance with ICH guidelines and was defined as 90-110% recovery of the initial concentration. Physical stability (color, pH, particulate matter, crystallization, and homogeneity) and microbiological stability were also evaluated. Results: The HPLC method demonstrated excellent linearity, precision, and accuracy. Oseltamivir concentrations remained within the predefined acceptance limits throughout the 75-day study period under freezing conditions, with no significant changes in pH, color, or particulate formation. After thawing, the drug concentration continued to remain fully stable and within the required limits for up to 10 days under refrigerated conditions. Despite the prolonged storage and phase changes, no significant changes in physical parameters were observed, and microbiological testing confirmed the absence of aerobic, anaerobic, and fungal microorganisms on the final day of the study. Conclusions: Oseltamivir 15 mg/mL oral solution in amber glass bottles is physicochemically and microbiologically stable for up to 85 days (75 days under frozen conditions plus 10 days under refrigeration after thawing).

Antiviral treatment (ART) resistance is an emerging global phenomenon that reduces the effectiveness of antiviral therapies and raises strong challenges for public health systems. This literature review focuses on the factors that contribute to developing antiviral drug resistance and the scope of pharmacogenetics. Studies have shown that inefficient use of antiviral therapies, genetic mutations in virus strains, and a limited healthcare framework are the main factors that increase resistance rates. Specifically, the poor adherence in HIV patients, combined with the virus high mutation, are the primary driver of drug-resistant strains development. The mutations in NS5A region reduce the efficacy of HCV direct-acting antivirals (DAAs). Where Influenza virus resistance to neuraminidase inhibitors (NAIs) like oseltamivir has been identified in seasonal strains, most notably the H275Y mutation in H1N1. Consequently, development of these resistant strains demands multidisciplinary strategies, global coordination, effective resistance monitoring systems, and public health initiatives to overcome this phenomenon. The paper aims to present a full picture of the antiviral drug resistance, which may provide better insights for clinicians and researchers.

Influenza antivirals (IAs) have been detected in aquatic environments inhabited by dabbling ducks, the natural reservoir of influenza A virus (IAV), raising concerns about the development of antiviral resistance. Because novel human IAV strains often contain genetic material of avian origin, this may contribute to resistance in viruses with pandemic potential. This study aimed to assess the environmental risk posed by four IAs-oseltamivir carboxylate (OC), zanamivir (ZA), peramivir (PE), and amantadine (AM)-based on their potential for environmental release, environmental stability, and induction of antiviral resistance. The assessment combined data from new experiments on (1) environmental release and (2) environmental stability of PE, AM, OC, and ZA, with results from previously published in vivo experiments in a mallard model examining (3) resistance development to OC, PE, and ZA in IAV. The risk of environmental release was assessed as high for OC, AM, and PE, and very high for ZA. Environmental stability ranged from very high to low, in the order PE > AM > OC > ZA. The potential to induce resistance in IAV was similar for PE and OC, and lower for ZA. Overall, the environmental risk ranking was PE > OC > ZA, with PE and OC posing the highest risks. Prudent use of IAs requires balancing the risk of resistance development against clinical benefit. In cases of complicated influenza or in high-risk patient groups, the clinical benefits are substantial and justify IA use. However, in uncomplicated influenza among otherwise healthy individuals, the clinical benefit is limited, and the risk of resistance development should be carefully considered. Among the evaluated antivirals, ZA showed the lowest environmental risk and should be preferred when feasible.

The extensive use of antiviral drugs for COVID-19 and influenza has increased their release into clinical and environmental settings, yet their potential to promote bacterial antibiotic cross-resistance remains poorly understood. Here, we investigated the antibacterial off-target effects of four clinically used anti-IFV/COV drugs-baloxavir acid (BXA), favipiravir (FAV), nirmatrelvir/ritonavir (NMV/r), and oseltamivir phosphate (OSV)-and their potential to influence bacterial stress responses and antibiotic cross-resistance in Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa PAO1. BXA and NMV/r showed clear antibacterial off-target effects, while antiviral exposure induced rapid oxidative and metabolic stress in a drug- and species-dependent manner. Following high-dose single-drug selection, evolved populations developed distinct antibiotic cross-resistance profiles, with BXA producing the broadest effects and B. subtilis showing the most recurrent β-lactam-associated responses. Under chronic environmental mixture exposure, low-dose antivirals further promoted β-lactam resistance, again with the strongest response observed in B. subtilis, highlighting the potential risk of antiviral residues as environmental selective pressures for antibiotic resistance. Comparative transcriptomic analysis further revealed that cross-resistance did not arise from a universal resistance pathway, but from drug-specific adaptive remodeling, including transporter and metabolic responses under BXA, sporulation/biofilm-associated developmental responses under NMV/r, and a more limited response to OSV. Together, this study provides initial systematic evidence that clinically used anti-IFV/COV drugs can directly perturb bacterial physiology and promote antibiotic cross-resistance under both elevated and environmentally relevant exposure scenarios, identifying antiviral contamination as an underappreciated driver of antibiotic resistance evolution.

Continuous monitoring of influenza antigenic drift is vital for vaccine strain selection. This study utilized global Department of War (DOW) surveillance data to evaluate circulating influenza strains and inform the 2026-2027 Northern Hemisphere influenza vaccine recommendations by the World Health Organization. From September 2025 through February 2026, the Department of Defense Global Respiratory Pathogen Surveillance Program collected and analyzed respiratory specimens from over 100 sentinel sites globally. Influenza-positive samples underwent next-generation sequencing, phylogenetic analysis, and antigenic characterization. Analysis of 1,300 sequences revealed A(H3N2) as the predominant (80.1%) subtype, followed by A(H1N1)pdm09 (10.6%) and B/Victoria (9.3%), with variations between combatant commands. Significant antigenic drift led to the dominance of subclades K, D.3.1/D.3.1.1, and C.3.1, respectively. Testing indicated that the 2025-2026 vaccine components offered reduced protection against these emerging strains. Antigenic cartography confirms that the 2026-2027 vaccine strains provide significantly improved protection against circulating viruses. Antiviral resistance markers for neuraminidase inhibitors such as oseltamivir remain rare, with only 2 identified in A(H1N1)pdm09. These data are critical for maintaining force health protection and assessing vaccine performance within highly mobile DOW populations. Analysis of 2025-2026 influenza season data revealed the predominance of subclades A(H1N1)pdm09 D.3.1/D.3.1.1, A(H3N2) K, and B/Victoria C.3.1. Antigenic cartography employing recent candidate vaccine viruses from the U.S. Centers for Disease Control and Prevention confirmed that the World Health Organization's strain recommendations for the 2026-2027 Northern Hemisphere influenza vaccine provide the best protection against these circulating strains.

The influenza A virus has been detrimental to public health for centuries and continues to take the lives of tens of thousands of people annually. Although current therapeutics have been effective in the past, the emergence of drug-resistant strains underscores the need for agents with new mechanisms of action. Here, we report thieno-[3,2-b]-pyrroles as novel group 2 influenza A virus entry inhibitors. In this study, we used a HA-based surrogate entry assay to perform the structure-activity relationship (SAR) studies to optimize the lead compounds. Docking studies in the predicted hemagglutinin binding site provided insight into potential binding interactions. Mutagenesis studies supported the proposed binding site. We validated the lead compounds that were potent in pseudovirus against infectious H3N2 strains including an oseltamivir-resistant strain. Lead compounds were counter-screened against other pseudoviruses and found to be selective. Together, these findings establish thieno-[3,2-b]-pyrroles as a promising new scaffold for combating group 2 influenza A viruses.

In 2023/24, A(H1N1)pdm09 influenza viruses with neuraminidase (NA)-S247N emerged in NA-clade C.5.3.3 carrying NA-I223V, spread internationally, then faded. Such double mutants reappeared sporadically in 2024/25. They expanded again in 2025/26 in NA-clade D.3 viruses carrying NA-S247N after acquiring NA-I223V in Europe - notably Spain, the Netherlands, Finland and France, and beyond. Dutch double mutants from both seasons showed median 12- and 13-fold reduced inhibition by oseltamivir. These findings underscore the need for ongoing genomic and phenotypic monitoring of antiviral susceptibility.

Seasonal influenza viruses continue to pose a significant threat to human health. As influenza viruses exhibit sustained genetic drift, it is imperative that animal studies utilize challenge strains that reflect contemporary, currently circulating viruses when evaluating pathogenicity, viral tropism, transmissibility, and antiviral sensitivity to better inform public health responses. Ferrets are considered the gold-standard small animal model for assessing currently circulating influenza viruses. Seasonal influenza A(H1N1)pdm09 viruses replicate well in both the upper and lower respiratory tract of ferrets, providing an important model for developing improved vaccination and therapeutic strategies; however, many of these studies have relied on a 2009 virus isolate. Utilising representative influenza A(H1N1)pdm09 virus strains from 2009 to 2022, we explored virus replication kinetics and lung pathogenesis in ferrets following intranasal inoculation with these contemporary strains. Our results revealed strain specific differences, with greater lung viral loads and pathogenesis following inoculation with A/Sydney/5/2021 compared to other strains. Efficient transmissibility of A/Sydney/5/2021 virus to naïve recipients was also observed following both contact and airborne exposure to infected donor ferrets. To refine this updated model, we performed side-by-side evaluation of oseltamivir antiviral efficacy following traditional intranasal or aerosol inhalation influenza challenges. Pre-treatment with oseltamivir demonstrated greater reductions in viral shedding from the upper respiratory tract than post-infection treatment of ferrets infected by aerosol inhalation, while the intranasal route showed reduced oseltamivir efficacy independent of the timing of antiviral treatment. These findings provide the basis for using an updated A(H1N1)pdm09 challenge virus for ferret studies as an alternative to the commonly used, but now less relevant 2009 early pandemic viruses. It also highlights how different methods of virus inoculation can influence outcomes of ferret antiviral studies, with an aerosol challenge model able to demonstrate differences between therapeutic and pre-treatment schedules, which were not apparent with an intranasal challenge model.