Acquisition of Amantadine Resistance via M Gene Reassortment in Canine H3N2 Influenza Virus and Elucidation of the Resistance Mechanism

Acquisition of Amantadine Resistance via M Gene Reassortment in Canine H3N2 Influenza Virus and Elucidation of the Resistance Mechanism | 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 Research Article Acquisition of Amantadine Resistance via M Gene Reassortment in Canine H3N2 Influenza Virus and Elucidation of the Resistance Mechanism Eulhae Ga, Eunseo Bae, Xing Xie, Jaehyun Hwang, Minjoo Yeom, Jong-woo Lim, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8173350/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Virology Journal → Version 1 posted 9 You are reading this latest preprint version Abstract A novel variant of the canine influenza virus H3N2 (cH3N2), designated as the M variant, was identified to contain a matrix (M) gene segment derived from the 2009 pandemic H1N1 virus (pdmH1N1), raising concerns regarding potential changes in antiviral drug sensitivity. In vitro and in vivo antiviral susceptibility assays demonstrated that while the parental cH3N2 strain was sensitive to amantadine, the M variant had acquired resistance to this drug. In contrast, both strains remained susceptible to neuraminidase inhibitors such as oseltamivir and zanamivir. Comparative amino acid sequence analysis of the M2 protein identified a substitution, L22S, uniquely present in amantadine resistant strains. Structural modeling of the M2 ion channel also suggested that the amantadine resistance observed in the M variant results from conformational alterations that impede drug binding. Collectively, these findings indicate that genetic reassortment with pdmH1N1 confers amantadine resistance in cH3N2 through the L22S substitution in the M2 protein. The preserved susceptibility to neuraminidase inhibitors suggests that these agents remain effective alternatives for controlling resistant strains, emphasizing the importance of continued molecular surveillance and diversified antiviral strategies. Influenza A virus reassortment amantadine antiviral drug resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introductions The 2009 pandemic H1N1 influenza virus (pdmH1N1) emerged as a novel strain that caused the first influenza pandemic of the 21st century. The virus was first detected in San Diego, California, in late March 2009 and rapidly disseminated worldwide. On June 11, 2009, the World Health Organization (WHO) declared a pandemic owing to rapid global spread of the virus. By August 2010, approximately 18,500 laboratory-confirmed deaths had been reported across 214 countries ( 1 ). The canine H3N2 influenza virus (cH3N2) is an avian-origin influenza A virus that has successfully adapted to infect and transmit among dogs. First identified in South Korea in 2007, cH3N2 subsequently spread to several countries, including China, Thailand, and the United States, becoming a major concern in veterinary medicine ( 2 ). Since its introduction into the canine population, cH3N2 has undergone adaptive mutations that enhanced its replication efficacy and transmissibility in dogs ( 3 ). In Korea, a reassortant virus containing the matrix (M) gene from the pdmH1N1 virus was later reported. Notably, this cH3N2 virus harboring the pdmH1N1-derived M segment exhibited enhanced transmissibility in ferrets compared with the parental cH3N2 strain ( 4 ). The M gene of influenza A viruses plays a critical role in viral morphology, assembly, and budding. This segment encodes two proteins, M1 and M2, through alternative splicing. The M1 protein, the most abundant structural protein, forms a layer beneath the viral envelope, providing structural integrity and mediating interactions with viral ribonucleoproteins (vRNPs) and the cytoplasmic tails of envelope glycoproteins to facilitate virion assembly ( 5 ). The M2 protein functions as a proton-selective ion channel essential for viral uncoating during entry and for pH regulation within the trans-Golgi network during viral maturation. During viral entry, M2-mediated proton influx induces the dissociation of M1 from vRNPs, a key step in releasing the viral genome into the host cell cytoplasm ( 6 ). Owing to its relatively low mutation rate and indispensable functional roles, the M segment is highly conserved and serves as an attractive target for antiviral drug development. One such antiviral, amantadine, specifically targets the M2 ion channel of influenza A viruses. Amantadine inhibits proton transport through M2, thereby blocking endosomal acidification required for viral uncoating and subsequent replication. Consequently, the viral genome cannot be released, effectively halting infection ( 7 ). However, amantadine resistance has become widespread among pdmH1N1 and contemporary avian influenza virus isolates, posing a significant challenge to antiviral control strategies ( 8 , 9 ). Recent advances in computational structural biology, particularly the development of AlphaFold, have revolutionized protein structure prediction and interaction modeling. AlphaFold provides near-experimental accuracy in predicting three-dimensional protein conformations, enabling detailed analyses of viral protein structures even in the absence of crystallographic data. This technology has increasingly been applied to model interactions between viral proteins and antiviral compounds, facilitating the identification of resistance-associated conformational changes and aiding rational drug design ( 10 – 12 ). Incorporating such structure-based approaches allows a more precise understanding of how reassortment or mutation in viral genes, such as the M segment, can alter drug-binding properties and influence antiviral susceptibility. Therefore, in this study, we investigated whether reassortant canine influenza viruses carrying the pdmH1N1-derived M segment have acquired resistance to amantadine and sought to elucidate the underlying molecular mechanisms of this resistance. 2. Materials and Methods 2.1. Cells and Viruses Madin-Darby canine kidney (MDCK, KCLB No. 10034) cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were cultured at 37°C in a humidified incubator containing 5% CO 2 . MDCK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, NY, USA, Cat. No. 10-013-CV) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin (100 units/mL)-streptomycin (100 µg/mL). The following influenza A viruses were propagated in MDCK cells: pdmH1N1 (A/California/04/2009 (CA04)), parental cH3N2 (A/canine/Korea/01/2007), M variant cH3N2 (A/canine/Korea/mv1/2012), H5N1 (A/chicken/Korea/ES/03), H5N2 (A/aquatic bird/Korea/CN2/2009), and H9N2 (A/wild bird/South Korea/snu6/2018). 2.2. Viral RNA extraction Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany, Cat. No. 52904) according to the manufacturer’s instructions. Briefly, Buffer AVL was added to the virus-containing supernatant, followed by carrier RNA to enhance recovery. The lysate was mixed with ethanol and transferred onto a QIAamp spin column, where viral RNA bound to the silica membrane. After centrifugation at 6000 x g for 1 min, the membrane was sequentially washed with Buffers AW1 and AW2. Purified RNA was eluted in 50 µL of Buffer AVE and stored at -80°C until further use. 2.3. Sequencing analyses To confirm viral identity, each gene segment was amplified by one-step RT-PCR using the Qiagen OneStep RT-PCR kit (Qiagen, Hilden, Germany, Cat. No. 210212) with universal influenza primers ( 13 ). Amplified products were purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany, Cat. No. 28704) and subjected to commercial Sanger sequencing (Cosmo Genetech, Seoul, Korea). 2.4. Measurement of half-maximal inhibitory concentration (IC 50 ) The IC 50 of each antiviral compound was determined by treating virus-infected (0.001 multiplicity of infection (MOI)) MDCK cells with serial dilutions of the respective drugs in 96-well plates. After 3-day incubation, viral replication and cell viability were quantified based on cytopathic effect (CPE). And the antiviral drug’s dose-response curves were generated. The IC 50 values were determined by fitting the inhibition data to sigmoid dose-response equations using nonlinear regression analysis, and results were reported as the drug concentration required to achieve 50% inhibition of viral replication. The antiviral agents used in the experiments were Amantadine hydrochloride (Sigma-Aldrich, St. Louis, USA, Cat. No. A1260), Zanamivir hydrate (Tokyo Chemical Industry, Japan, Cat. No. Z0023), and Oseltamivir acid (Cayman Chemical, MI, USA, Cat. No. 15779). 2.5. Protein structure prediction and amantadine binding analysis using AlphaFold The tertiary structures of the influenza M2 proteins were predicted using the AlphaFold 3 platform ( https://alphafoldserver.com/ ). Amino acid sequences of the M2 proteins from both the parental cH3N2 and M variant cH3N2 viruses were modeled as tetrameric assemblies, reflecting their biologically functional configuration as proton-selective ion channels. The AlphaFold system employs deep learning-based algorithms trained on experimentally derived crystallographic and cryo-EM data to predict near-atomic resolution structures, allowing accurate inference of conformational features even in the absence of crystallographic data. In addition to structural prediction, the modeled M2 channel structures were further analyzed to investigate potential amantadine-binding sites and interaction patterns. Molecular docking simulations were subsequently performed to evaluate the potential interactions between the modeled M2 ion channel and amantadine. Docking and visualization were conducted using PyMOL Molecular Graphics System (Schrödinger LLC), and conformational differences between the parental and M variant M2 channels were analyzed to assess alterations potentially responsible for drug resistance. 2.6. Indirect immunofluorescence assay (IFA) MDCK cells were seeded at a density of 3 × 10 4 cells per well in 96-well plates and incubated until a confluent monolayer was formed. For viral infection, the cells were infected with 0.001 MOI of each virus in a volume of 100 µL per well and incubated for 1 h at 37°C in the presence of 100µM of the antiviral compounds. After infection, the inoculum was removed, and the cells were washed twice with PBS. Subsequently, serum-free DMEM containing 1 µg/mL TPCK-trypsin and the same concentration of the antiviral compound was added, followed by incubation for 12 h at 37°C. After incubation, the cells were fixed with 4% paraformaldehyde (GeneAll Biotechnology, Seoul, Korea, Cat. No. SM-P01-050) for 15 min, permeabilized with 0.1% Triton X-100 (Fisher BioReagents, Waltham, MA, USA, Cat. No. BP151) for 10 min, and blocked with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA, Cat. A8412) at room temperature for 1 h. After each step, the cells were washed with PBST (0.05% Tween 20). The cells were then incubated overnight at 4°C with anti-IAV NP antibody (1:500 dilution; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. PA5-32242). After washing three times with PBST (0.05% Tween 20), the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500 dilution; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. A-11008) for 1h at room temperature. Following secondary antibody incubation, the cells were washed three times with PBST, and nuclei were counterstained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. D21490). Fluorescence images were acquired using a digital inverted fluorescence microscope (DMi8; Leica Microsystems, Wetzlar, Germany). 2.7. Evaluation of antiviral efficacy in mice To evaluate the antiviral efficacy of amantadine, five-week-old female BALB/c mice (Koatech, Korea) were used. Mice were randomly assigned to experimental groups (n = 8 per virus strain). Amantadine hydrochloride was administered orally (p.o.) at a dose of 100 mg/kg/day, twice daily. The initial treatment was given 4 h before viral infection, followed by oral administrations every 12 h for 5 days. Mice were anesthetized and infected intranasally with 30 µL of viral inoculum (10⁷ TCID₅₀/mL) of either parental cH3N2 (A/canine/Korea/01/2007) or M variant cH3N2 (A/canine/Korea/mv1/2012). At 3 and 5days post infection (DPI), mice (n = 4 per group per time point) were euthanized, and the lungs were collected for viral quantification. The tissues were homogenized in PBS, and the homogenates were clarified by centrifugation at 3,000 × g for 10 min. The supernatants were used for viral RNA quantification. Viral RNA extraction and Reverse transcription quantitative PCR (RT-qPCR) targeting the M gene of influenza A virus was performed. RT-qPCR was performed using the Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 5555532) ( 14 ). All animal experiments were conducted at Seoul National University, Seoul, Korea, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (approval number SNU-250922-1), in accordance with institutional and national guidelines for animal care and use. 2.8. Statistical Analysis Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel. Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. For comparisons among multiple groups, two-way analysis of variance (ANOVA) was performed, followed by Tukey’s multiple-comparisons test to determine statistical significance. A p value of < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001). 3. Results 3.1. Susceptibility of viruses to amantadine Reverse zoonotic transmission and subsequent genetic reassortment were observed to give rise to influenza variants exhibiting antiviral resistance ( 15 ). The parental cH3N2 strain (A/canine/Korea/01/2007) was sensitive to amantadine, exhibiting an IC 50 value of 0.1 µM. In contrast, the M variant cH3N2 (A/canine/Korea/MV1/2012), the pdmH1N1 strain (A/California/04/2009), which served as the donor of the reassorted M gene, and other avian influenza strains (A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, and A/wild bird/South Korea/snu6/2018) exhibited marked resistance to amantadine even at the highest tested concentration of 1000 µM (Table 1 ). These findings indicate that the acquisition of the M gene from pdmH1N1 conferred amantadine resistance to the reassorted cH3N2 virus. 3.2. Susceptibility of viruses to oseltamivir and zanamivir It was found that the reassortment of the M gene did not affect the susceptibility of the virus to neuraminidase-targeting antiviral agents. Both the parental and M variant cH3N2 strains remained sensitive to oseltamivir, with IC 50 values of 0.002 µM and 0.005 µM, respectively. Similarly, both strains were sensitive to zanamivir, exhibiting an IC 50 values of 0.05 µM (Table 2 ). These results suggest that antiviral agents targeting proteins other than the M gene, such as neuraminidase inhibitors, retain efficacy regardless of M gene reassortment. Consistently, the pdmH1N1 and avian influenza strains also exhibited low IC 50 values against oseltamivir and zanamivir, confirming their maintained susceptibility to these drugs. Table 1 Half-maximal inhibitory concentration (IC 50 ) of influenza virus strains against amantadine Virus strain Amantadine IC 50 Amino Acid at Position 22 Response to Amantadine A/canine/Korea/01/2007 0.1µM Leucine (L) Sensitive A/canine/Korea/MV1/2012 > 1000µM Serine (S) Resistant A/California/04/2009 A/chicken/Korea/ES/03 A/aquatic bird/Korea/CN2/2009 A/wild bird/South Korea/snu6/2018 The parental cH3N2 (A/canine/Korea/01/2007) strain exhibited an IC₅₀ of 0.1 µM against amantadine, indicating sensitivity. In contrast, the M variant cH3N2 (A/canine/Korea/MV1/2012) and other strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018) displayed drug resistance, as no measurable IC₅₀ to the amantadine could be determined. Table 2 Half-maximal inhibitory concentration (IC 50 ) of influenza virus strains against oseltamivir and zanamivir Virus strain Oseltamivir IC 50 Response to Oseltamivir Zanamivir IC 50 Response to Zanamivir A/canine/Korea/01/2007 0.002µM Sensitive 0.05µM Sensitive A/canine/Korea/MV1/2012 0.005µM 0.05µM A/California/04/2009 0.02µM 0.05µM A/chicken/Korea/ES/03 0.005µM 0.05µM A/aquatic bird/Korea/CN2/2009 0.05µM 0.2µM A/wild bird/South Korea/snu6/2018 0.001µM 0.05µM The parental cH3N2 (A/canine/Korea/01/2007), the M variant cH3N2 (A/canine/Korea/MV1/2012), and other strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018) exhibited an IC₅₀ of 0.001–0.05 µM against oseltamivir and 0.05–0.2 µM against zanamivir, indicating moderate sensitivity to the neuraminidase-targeting antiviral agents. 3.3. Amino acid sequence comparison of the M2 protein To identify the molecular determinant responsible for amantadine resistance, the M2 protein sequences from the parental cH3N2, M variant cH3N2, and several amantadine-resistant strains were aligned and compared (Fig. 1 ). Multiple amino acid substitutions were observed across the M gene among these viruses. However, one specific position, amino acid residue 22, showed a unique pattern that strongly correlated with the antiviral phenotype. All amantadine-resistant viruses, including the M variant cH3N2, the pdmH1N1 strain, and the avian influenza strains, possessed a serine (S) residue at position 22, whereas the amantadine-sensitive parental cH3N2 uniquely retained a leucine (L) at this site. Although other substitutions were present, none showed a consistent difference between the resistant and sensitive groups. Therefore, residue 22 represents the only amino acid position that consistently distinguishes the amantadine-sensitive strain from all resistant strains, suggesting that the L22S substitution is the unique molecular determinant responsible for the observed amantadine resistance. This figure presents a comparison of amino acid sequences among various influenza virus strains, including parental cH3N2 (A/canine/Korea/01/2007), M variant cH3N2 (A/canine/Korea/MV1/2012), and other amantadine-resistant strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018). Notably, at position 22, only the parental cH3N2 strain possesses a leucine (L) residue, whereas all other strains have a serine (S) at this position. This unique substitution is exclusive to the parental cH3N2. These results suggest that the amino acid variation at position 22 may contribute to the distinct characteristics of the amantadine resistance. 3.4. Predicted structural conformations and interactions of the M2 protein with amantadine To further investigate this hypothesis, we employed AlphaFold to predict the structural conformations of the M2 protein channels and their potential interactions with amantadine (Fig. 2 ). Because neuraminidase-targeting antiviral agents do not interact with the M2 protein, interaction analysis was performed only between amantadine and the M2 protein of the parental cH3N2 and pdmH1N1 viruses. A structural comparison of the amino acid at position 22 revealed that in the parental cH3N2 virus, the leucine residue projects into the channel lumen, where it interacts with amantadine and contributes to the blockage of the channel. In contrast, in the resistant strains, the serine residue moves away from the channel lumen and is buried within the structure, resulting in an expanded channel that prevents amantadine from blocking the M2 ion channel, which is the mechanism of its antiviral activity. 3.5. Immunofluorescence assay (IFA) To further assess the antiviral effects of amantadine, oseltamivir, and zanamivir, MDCK cells were infected with the same panel of influenza virus strains described above and treated with 100 µM of each compound for 12 h. Viral NP protein expression was examined by fluorescence microscope. In the cells infected with the viruses without drug treatment, strong NP fluorescence was observed, confirming successful viral infection. In contrast, NP expression was completely suppressed in parental cH3N2 infected cells treated with amantadine, with no detectable green fluorescence. However, cells infected with the M variant cH3N2, pdmH1N1, or avian influenza strains retained strong NP fluorescence despite amantadine treatment, indicating resistance to the drug. Treatment with oseltamivir or zanamivir resulted in a loss of NP fluorescence across all virus groups, consistent with inhibition of viral replication (Fig. 3 ). AlphaFold-predicted structures of the M2 ion channel from the parental cH3N2 virus (with leucine at position 22) and the human pandemic H1N1 virus (with serine at position 22) were shown. In the parental cH3N2 structure, the leucine residue (red sticks) at position 22 projects into the channel lumen, where it directly interacts with amantadine (orange cubic structure) and contributes to the effective blockage of the channel. In contrast, in the pandemic H1N1 structure, the serine residue (blue sticks) at the same position is oriented away from the channel lumen and is buried within the structure, resulting in an expanded channel. This conformational difference prevents amantadine from blocking the M2 ion channel in the pandemic H1N1 strain. MCDK cells were infected with each influenza virus strain at 0.001 MOI, and viral NP protein expression was detected by IFA at 12 hours post-infection using an anti-NP antibody (green). The nucleus was counterstained with DAPI (blue). In cells infected with the parental cH3N2 strain and treated with amantadine, NP expression was completely suppressed, and no green fluorescence was detected. However, in the M variant cH3N2, pandemic H1N1, and avian influenza strains, strong cytoplasmic NP fluorescence persisted despite amantadine treatment, demonstrating resistance to the drug. Oseltamivir and zanamivir treatment led to marked reduction or complete loss of NP fluorescence across all virus groups, consistent with inhibition of viral replication. 3.6. In vivo antiviral efficacy of amantadine To evaluate the in vivo antiviral efficacy of amantadine, five-week-old female BALB/c mice were intranasally infected with either the parental or M variant cH3N2 virus, followed by oral administration of amantadine (100 mg/kg/day, twice daily) starting 4 h before infection and continuing every 12 h for 5 days. The virus control group, which served as the positive control, consisted of infected mice treated with PBS instead of amantadine. Lungs were collected at DPI 3 and 5. At DPI 3, amantadine treated group infected with the parental cH3N2 virus exhibited alleviated gross lung lesions characterized by lighter coloration and reduced consolidation compared with the virus control group. In contrast, lungs from the virus control group appeared dark red to brown, indicative of viral pneumonia and severe inflammation. However, no apparent differences were observed between amantadine treated and virus control groups in mice infected with the M variant cH3N2 virus, suggesting a loss of therapeutic efficacy in the M gene–reassorted strain (Fig. 4 ). Consistent with these pathological findings, RT-qPCR analysis targeting the M gene revealed a significant reduction in viral RNA levels in amantadine treated mice infected with the parental cH3N2 strain. (Fig. 5 ). At DPI 3, viral RNA levels (expressed as 40 – Cq values) were markedly lower in the treated group than in the untreated controls (p < 0.001) (Fig. 5 A), and the difference remained significant at DPI 5 (p 0.05 at both DPI 3 and 5). At DPI 3, amantadine treatment induced a significantly greater reduction of viral RNA in the parental cH3N2 group than in the M variant group (p < 0.01), indicating that the parental strain was more susceptible to the antiviral effect of amantadine. However, no significant difference between the two strains was observed at DPI 5 (Fig. 5 C). These results collectively demonstrate that amantadine effectively inhibits viral replication and mitigates lung pathology in mice infected with the parental cH3N2 virus, but fails to confer antiviral protection against the M gene-reassorted variant, highlighting the impact of M2 mutations on amantadine resistance. Representative lungs collected at DPI 3 from mice infected with either the parental or M variant cH3N2 virus. Amantadine treatment visibly reduced pulmonary lesions in parental cH3N2 infected mice, but not in M variant cH3N2 infected mice. Viral RNA levels in lung homogenates were quantified by RT-qPCR targeting the M gene at DPI 3 and 5. (A and B) Amantadine treatment reduced viral RNA levels at DPI 3 and DPI 5 in parental cH3N2 infected mice, but not in M variant cH3N2 infected mice. Viral RNA reductions in the lungs of amantadine-treated mice were expressed as log₂-transformed values. (C) At DPI 3, amantadine treatment induced a significantly greater reduction of viral RNA in the parental cH3N2 group compared with the M variant group (p < 0.01), whereas no significant difference was observed at DPI 5. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. Data represent mean ± SD. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. 4. Discussion In this study, we first demonstrated that canine influenza viruses acquiring the pdmH1N1-derived M segment through reassortment can subsequently showed resistance to amantadine. Furthermore, by comparing the amino acid sequences of amantadine-resistant and -sensitive strains, we identified the specific residues responsible for resistance acquisition. Structural modeling analysis further elucidated the underlying mechanism by which these mutations confer resistance. We figured out acquired amantadine resistance of reassortant canine influenza viruses carrying the pdmH1N1 M segment and proposed alternative antiviral drugs. In addition, by comparing the amino acid sequences of the M2 protein between amantadine-susceptible and -resistant strains followed by structural analysis, we identified a key substitution associated with resistance and demonstrated how this mutation affects drug efficacy. Genetic reassortment, a common mechanism of influenza virus evolution, led to the emergence of a variant strain that exhibited resistance to amantadine. This phenotypic shift highlights the potential for rapid adaptation of influenza viruses under antiviral pressure, and underscores the importance of continuous molecular surveillance. Sequence alignment of the M2 protein across susceptible and resistant strains revealed a consistent amino acid substitution at position 22, where leucine (L) in the parental cH3N2 was replaced by serine (S) in resistant strains. Notably, this L22S mutation was the only consistent difference in M2 protein sequences correlating with the resistance phenotype, suggesting a causative role in mediating amantadine resistance. Structural modeling using AlphaFold provided mechanistic insights into the functional impact of this substitution. In the susceptible virus, the leucine residue at position 22 protrudes into the M2 channel lumen, where it appears to directly participate in amantadine binding and the subsequent inhibition of ion conductance. In contrast, in the resistant strains, the serine residue is structurally repositioned away from the lumen and buried within the transmembrane domain, leading to a widened channel conformation that likely impairs amantadine binding and its inhibitory function. This structural alteration provides a plausible mechanistic explanation for the observed loss of drug efficacy. While the current findings clearly demonstrate the structural and phenotypic basis of amantadine resistance, several experimental limitations should be acknowledged. The influenza virus strains used in this study exhibited relatively low pathogenicity in mice, preventing accurate assessment of survival rates following infection. Instead, viral replication in the lungs was quantified as a surrogate marker of in vivo antiviral efficacy, which still provided a reliable measure of drug responsiveness. Moreover, reverse genetics-based point-mutation viruses were not generated to experimentally confirm the causal role of the identified M2 substitution. Nevertheless, the use of naturally occurring M variant isolated offers clinically meaningful evidence, as these field-derived viruses represent authentic resistance phenotypes circulating in nature. Despite the acquired resistance to amantadine, the M variant cH3N2 retained susceptibility to neuraminidase (NA)-targeting antiviral agents, including oseltamivir and zanamivir. This observation is consistent with the conservation of the NA protein sequence and function, and suggests that these NA inhibitors remain viable treatment options in the context of M2-targeted drug resistance. These findings support the potential utility of combination antiviral therapy or NA inhibitor monotherapy as alternative strategies in cases where amantadine resistance is detected. Taken together, this study not only identifies a specific amino acid mutation responsible for amantadine resistance in reassortant influenza viruses, but also demonstrates how structural modeling can complement sequence-based analyses to elucidate resistance mechanisms. Moreover, the maintenance of NA inhibitor susceptibility offers a practical therapeutic alternative and emphasizes the importance of multi-targeted antiviral development. Continued surveillance for resistance mutations and further functional validation of these findings in vivo will be essential for guiding future antiviral strategies against evolving influenza viruses. 5. Conclusion This study demonstrated that resistance to amantadine can be acquired through reassortment with pandemic influenza viruses. Sequence and structural analyses identified a single M2 substitution, L22S, as the key determinant responsible for this resistance, providing a clear mechanistic explanation for impaired drug binding. Despite the acquisition of amantadine resistance, the retained susceptibility to NA inhibitors supports their continued usefulness as effective therapeutic options for controlling resistant variant strains. These finding highlight the evolutionary potential of influenza viruses to rapidly alter antiviral susceptibility and emphasize the need for ongoing molecular surveillance and diversified antiviral strategies. Abbreviations pdmH1N1: 2009 pandemic H1N1 influenza virus cH3N2: Canine H3N2 influenza virus M1: Matrix protein 1 M2: Matrix protein 2 CPE: Cytopathic effect IC 50 : Half-maximal inhibitory concentration MOI: Multiplicity of infection TCID 50 : 50% tissue culture infectious dose IFA: Indirect immunofluorescence assay NP: Nucleoprotein NA: Neuraminidase DPI: Day post infection Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (approval number SNU-250922-1). All procedures involving animals were conducted in accordance with the institutional guidelines for the care and use of laboratory animals and the national regulations of the Republic of Korea. Five-week-old female BALB/c mice (Koatech, Korea) were housed under specific-pathogen-free (SPF) conditions, and appropriate anesthesia and humane euthanasia procedures were employed to minimize animal suffering. Consent for publication Not applicable Availability of data and materials The manuscript includes all datasets generated or analyzed during this study. Data will be made available on request. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through, High-Risk Animal infectious Disease Control Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (RS-2025-02304688) and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2024-00432287). Authors’ contributions E.G., E.B., X.X., and W.N. conceived and wrote the manuscript, E.G., X.X., M.Y., and D.S. contributed on in vitro experiment, E.G.,and E.B. contributed on in vivo experiment, J.H., J.-W.L. and W.N. made the figures, E.G., X.X., D.S., and W.N. reviewed and edited the manuscript. All authors read and approved the final manuscript. Author’s information Eulhae Ga, Eunseo Bae and Xing xie contributed equally to this work. Corresponding author Correspondence to Daesub Song ( [email protected] ) and Woonsung Na ( [email protected] ) References Gaitonde DY, Moore FC, Morgan MK. Influenza: diagnosis and treatment. Am Family Phys. 2019;100(12):751–8. Song D, Kang B, Lee C, Jung K, Ha G, Kang D, et al. Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis. 2008;14(5):741. Song D, Moon H-J, An D-J, Jeoung H-Y, Kim H, Yeom M-J, et al. A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea. J Gen Virol. 2012;93(3):551–4. Moon H, Hong M, Kim J, Seon B, Na W, Park S-J, et al. H3N2 canine influenza virus with the matrix gene from the pandemic A/H1N1 virus: infection dynamics in dogs and ferrets. Epidemiol Infect. 2015;143(4):772–80. Bui M, Wills EG, Helenius A, Whittaker GR. Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol. 2000;74(4):1781–6. Pielak RM, Chou JJ. Influenza M2 proton channels. Biochim et Biophys Acta (BBA)-Biomembranes. 2011;1808(2):522–9. Hay A, Wolstenholme A, Skehel J, Smith MH. The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 1985;4(11):3021–4. Cho H-G, Choi J-H, Kim W-H, Hong H-K, Yoon M-H, Jho E-H, et al. High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005–2010. Arch Virol. 2013;158:241–5. Zaraket H, Saito R, Suzuki Y, Baranovich T, Dapat C, Caperig-Dapat I, et al. Genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza A/H1N1 viruses. J Clin Microbiol. 2010;48(4):1085–92. Wong F, Krishnan A, Zheng EJ, Stärk H, Manson AL, Earl AM, et al. Benchmarking AlphaFold-enabled molecular docking predictions for antibiotic discovery. Mol Syst Biol. 2022;18(9):e11081. Gutnik D, Evseev P, Miroshnikov K, Shneider M. Using AlphaFold predictions in viral research. Curr Issues Mol Biol. 2023;45(4):3705–32. Desai D, Kantliwala SV, Vybhavi J, Ravi R, Patel H, Patel J. Review of AlphaFold 3: transformative advances in drug design and therapeutics. Cureus. 2024;16(7):e63646. Hoffmann E, Stech J, Guan Y, Webster R, Perez D. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol. 2001;146:2275–89. Chen Y, Cui D, Zheng S, Yang S, Tong J, Yang D, et al. Simultaneous detection of influenza A, influenza B, and respiratory syncytial viruses and subtyping of influenza A H3N2 virus and H1N1 (2009) virus by multiplex real-time PCR. J Clin Microbiol. 2011;49(4):1653–6. Nelson MI, Vincent AL. Reverse zoonosis of influenza to swine: new perspectives on the human–animal interface. Trends Microbiol. 2015;23(3):142–53. Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx Cite Share Download PDF Status: Published Journal Publication published 13 Feb, 2026 Read the published version in Virology Journal → Version 1 posted Editorial decision: Revision requested 09 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviewers invited by journal 25 Nov, 2025 Editor assigned by journal 24 Nov, 2025 Submission checks completed at journal 24 Nov, 2025 First submitted to journal 21 Nov, 2025 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":469571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmino acid alignment of the M2 protein from parental cH3N2, M variant cH3N2, and amantadine-resistant viruses\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/3247acf76e0cdd7b7cc1b9db.png"},{"id":97010089,"identity":"8c1bf03a-e241-4f1e-8b95-cab3e7687337","added_by":"auto","created_at":"2025-11-28 15:30:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":469562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePredicted structural conformations of the M2 protein channels and their interactions with amantadine using AlphaFold\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/7700a7208f84680807e9bca3.png"},{"id":97137838,"identity":"68c8b1dd-4383-48b0-8fd5-20cbbad1c5a2","added_by":"auto","created_at":"2025-12-01 09:58:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":401203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence assay (IFA) of influenza viral NP expression in MDCK cells infected with various influenza virus strains and treated with different antiviral agents.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/25e94bcc3f8de41a959be08e.png"},{"id":97138828,"identity":"0b1b2f4b-e647-4955-9f99-9d17bc2e4806","added_by":"auto","created_at":"2025-12-01 09:59:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGross lung morphology of amantadine-treated and untreated mice infected with cH3N2 viruses.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/57b799087571e32d5b3a01f0.png"},{"id":97139291,"identity":"643f1c54-7855-4b8c-8799-2407add91e42","added_by":"auto","created_at":"2025-12-01 09:59:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntiviral effects of amantadine in mice infected with parental or M variant cH3N2 viruses.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/9abb75ca0670505f74b4cfda.png"},{"id":102785587,"identity":"76ecd542-7c68-4d30-b3d4-c76704209e70","added_by":"auto","created_at":"2026-02-16 16:08:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2592227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/57cf38bc-d422-4af2-a22c-53ffaa8b1521.pdf"},{"id":97139506,"identity":"21de424c-4360-4dcc-b4d9-14d23ff46b1b","added_by":"auto","created_at":"2025-12-01 10:00:32","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2894443,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8173350/v1/fa67b68d11563c9a0230e9be.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acquisition of Amantadine Resistance via M Gene Reassortment in Canine H3N2 Influenza Virus and Elucidation of the Resistance Mechanism","fulltext":[{"header":"1. Introductions","content":"\u003cp\u003eThe 2009 pandemic H1N1 influenza virus (pdmH1N1) emerged as a novel strain that caused the first influenza pandemic of the 21st century. The virus was first detected in San Diego, California, in late March 2009 and rapidly disseminated worldwide. On June 11, 2009, the World Health Organization (WHO) declared a pandemic owing to rapid global spread of the virus. By August 2010, approximately 18,500 laboratory-confirmed deaths had been reported across 214 countries (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe canine H3N2 influenza virus (cH3N2) is an avian-origin influenza A virus that has successfully adapted to infect and transmit among dogs. First identified in South Korea in 2007, cH3N2 subsequently spread to several countries, including China, Thailand, and the United States, becoming a major concern in veterinary medicine (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Since its introduction into the canine population, cH3N2 has undergone adaptive mutations that enhanced its replication efficacy and transmissibility in dogs (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In Korea, a reassortant virus containing the matrix (M) gene from the pdmH1N1 virus was later reported. Notably, this cH3N2 virus harboring the pdmH1N1-derived M segment exhibited enhanced transmissibility in ferrets compared with the parental cH3N2 strain (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe M gene of influenza A viruses plays a critical role in viral morphology, assembly, and budding. This segment encodes two proteins, M1 and M2, through alternative splicing. The M1 protein, the most abundant structural protein, forms a layer beneath the viral envelope, providing structural integrity and mediating interactions with viral ribonucleoproteins (vRNPs) and the cytoplasmic tails of envelope glycoproteins to facilitate virion assembly (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The M2 protein functions as a proton-selective ion channel essential for viral uncoating during entry and for pH regulation within the trans-Golgi network during viral maturation. During viral entry, M2-mediated proton influx induces the dissociation of M1 from vRNPs, a key step in releasing the viral genome into the host cell cytoplasm (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOwing to its relatively low mutation rate and indispensable functional roles, the M segment is highly conserved and serves as an attractive target for antiviral drug development. One such antiviral, amantadine, specifically targets the M2 ion channel of influenza A viruses. Amantadine inhibits proton transport through M2, thereby blocking endosomal acidification required for viral uncoating and subsequent replication. Consequently, the viral genome cannot be released, effectively halting infection (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, amantadine resistance has become widespread among pdmH1N1 and contemporary avian influenza virus isolates, posing a significant challenge to antiviral control strategies (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent advances in computational structural biology, particularly the development of AlphaFold, have revolutionized protein structure prediction and interaction modeling. AlphaFold provides near-experimental accuracy in predicting three-dimensional protein conformations, enabling detailed analyses of viral protein structures even in the absence of crystallographic data. This technology has increasingly been applied to model interactions between viral proteins and antiviral compounds, facilitating the identification of resistance-associated conformational changes and aiding rational drug design (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Incorporating such structure-based approaches allows a more precise understanding of how reassortment or mutation in viral genes, such as the M segment, can alter drug-binding properties and influence antiviral susceptibility.\u003c/p\u003e\u003cp\u003eTherefore, in this study, we investigated whether reassortant canine influenza viruses carrying the pdmH1N1-derived M segment have acquired resistance to amantadine and sought to elucidate the underlying molecular mechanisms of this resistance.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cells and Viruses\u003c/h2\u003e\u003cp\u003eMadin-Darby canine kidney (MDCK, KCLB No. 10034) cells were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cells were cultured at 37\u0026deg;C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e. MDCK cells were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Corning, NY, USA, Cat. No. 10-013-CV) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin (100 units/mL)-streptomycin (100 \u0026micro;g/mL). The following influenza A viruses were propagated in MDCK cells: pdmH1N1 (A/California/04/2009 (CA04)), parental cH3N2 (A/canine/Korea/01/2007), M variant cH3N2 (A/canine/Korea/mv1/2012), H5N1 (A/chicken/Korea/ES/03), H5N2 (A/aquatic bird/Korea/CN2/2009), and H9N2 (A/wild bird/South Korea/snu6/2018).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Viral RNA extraction\u003c/h2\u003e\u003cp\u003eViral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany, Cat. No. 52904) according to the manufacturer\u0026rsquo;s instructions. Briefly, Buffer AVL was added to the virus-containing supernatant, followed by carrier RNA to enhance recovery. The lysate was mixed with ethanol and transferred onto a QIAamp spin column, where viral RNA bound to the silica membrane. After centrifugation at 6000 x g for 1 min, the membrane was sequentially washed with Buffers AW1 and AW2. Purified RNA was eluted in 50 \u0026micro;L of Buffer AVE and stored at -80\u0026deg;C until further use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Sequencing analyses\u003c/h2\u003e\u003cp\u003eTo confirm viral identity, each gene segment was amplified by one-step RT-PCR using the Qiagen OneStep RT-PCR kit (Qiagen, Hilden, Germany, Cat. No. 210212) with universal influenza primers (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Amplified products were purified with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany, Cat. No. 28704) and subjected to commercial Sanger sequencing (Cosmo Genetech, Seoul, Korea).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Measurement of half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e)\u003c/h2\u003e\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e of each antiviral compound was determined by treating virus-infected (0.001 multiplicity of infection (MOI)) MDCK cells with serial dilutions of the respective drugs in 96-well plates. After 3-day incubation, viral replication and cell viability were quantified based on cytopathic effect (CPE). And the antiviral drug\u0026rsquo;s dose-response curves were generated. The IC\u003csub\u003e50\u003c/sub\u003e values were determined by fitting the inhibition data to sigmoid dose-response equations using nonlinear regression analysis, and results were reported as the drug concentration required to achieve 50% inhibition of viral replication. The antiviral agents used in the experiments were Amantadine hydrochloride (Sigma-Aldrich, St. Louis, USA, Cat. No. A1260), Zanamivir hydrate (Tokyo Chemical Industry, Japan, Cat. No. Z0023), and Oseltamivir acid (Cayman Chemical, MI, USA, Cat. No. 15779).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Protein structure prediction and amantadine binding analysis using AlphaFold\u003c/h2\u003e\u003cp\u003eThe tertiary structures of the influenza M2 proteins were predicted using the AlphaFold 3 platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafoldserver.com/\u003c/span\u003e\u003cspan address=\"https://alphafoldserver.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Amino acid sequences of the M2 proteins from both the parental cH3N2 and M variant cH3N2 viruses were modeled as tetrameric assemblies, reflecting their biologically functional configuration as proton-selective ion channels. The AlphaFold system employs deep learning-based algorithms trained on experimentally derived crystallographic and cryo-EM data to predict near-atomic resolution structures, allowing accurate inference of conformational features even in the absence of crystallographic data. In addition to structural prediction, the modeled M2 channel structures were further analyzed to investigate potential amantadine-binding sites and interaction patterns. Molecular docking simulations were subsequently performed to evaluate the potential interactions between the modeled M2 ion channel and amantadine. Docking and visualization were conducted using PyMOL Molecular Graphics System (Schr\u0026ouml;dinger LLC), and conformational differences between the parental and M variant M2 channels were analyzed to assess alterations potentially responsible for drug resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Indirect immunofluorescence assay (IFA)\u003c/h2\u003e\u003cp\u003eMDCK cells were seeded at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates and incubated until a confluent monolayer was formed. For viral infection, the cells were infected with 0.001 MOI of each virus in a volume of 100 \u0026micro;L per well and incubated for 1 h at 37\u0026deg;C in the presence of 100\u0026micro;M of the antiviral compounds. After infection, the inoculum was removed, and the cells were washed twice with PBS. Subsequently, serum-free DMEM containing 1 \u0026micro;g/mL TPCK-trypsin and the same concentration of the antiviral compound was added, followed by incubation for 12 h at 37\u0026deg;C. After incubation, the cells were fixed with 4% paraformaldehyde (GeneAll Biotechnology, Seoul, Korea, Cat. No. SM-P01-050) for 15 min, permeabilized with 0.1% Triton X-100 (Fisher BioReagents, Waltham, MA, USA, Cat. No. BP151) for 10 min, and blocked with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA, Cat. A8412) at room temperature for 1 h. After each step, the cells were washed with PBST (0.05% Tween 20). The cells were then incubated overnight at 4\u0026deg;C with anti-IAV NP antibody (1:500 dilution; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. PA5-32242). After washing three times with PBST (0.05% Tween 20), the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500 dilution; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. A-11008) for 1h at room temperature. Following secondary antibody incubation, the cells were washed three times with PBST, and nuclei were counterstained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA, Cat. No. D21490). Fluorescence images were acquired using a digital inverted fluorescence microscope (DMi8; Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Evaluation of antiviral efficacy in mice\u003c/h2\u003e\u003cp\u003eTo evaluate the antiviral efficacy of amantadine, five-week-old female BALB/c mice (Koatech, Korea) were used. Mice were randomly assigned to experimental groups (n\u0026thinsp;=\u0026thinsp;8 per virus strain). Amantadine hydrochloride was administered orally (p.o.) at a dose of 100 mg/kg/day, twice daily. The initial treatment was given 4 h before viral infection, followed by oral administrations every 12 h for 5 days. Mice were anesthetized and infected intranasally with 30 \u0026micro;L of viral inoculum (10⁷ TCID₅₀/mL) of either parental cH3N2 (A/canine/Korea/01/2007) or M variant cH3N2 (A/canine/Korea/mv1/2012). At 3 and 5days post infection (DPI), mice (n\u0026thinsp;=\u0026thinsp;4 per group per time point) were euthanized, and the lungs were collected for viral quantification. The tissues were homogenized in PBS, and the homogenates were clarified by centrifugation at 3,000 \u0026times; g for 10 min. The supernatants were used for viral RNA quantification. Viral RNA extraction and Reverse transcription quantitative PCR (RT-qPCR) targeting the M gene of influenza A virus was performed. RT-qPCR was performed using the Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 5555532) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). All animal experiments were conducted at Seoul National University, Seoul, Korea, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (approval number SNU-250922-1), in accordance with institutional and national guidelines for animal care and use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments. For comparisons among multiple groups, two-way analysis of variance (ANOVA) was performed, followed by Tukey\u0026rsquo;s multiple-comparisons test to determine statistical significance. A \u003cem\u003ep\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant (*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Susceptibility of viruses to amantadine\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eReverse zoonotic transmission and subsequent genetic reassortment were observed to give rise to influenza variants exhibiting antiviral resistance (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The parental cH3N2 strain (A/canine/Korea/01/2007) was sensitive to amantadine, exhibiting an IC\u003csub\u003e50\u003c/sub\u003e value of 0.1 \u0026micro;M. In contrast, the M variant cH3N2 (A/canine/Korea/MV1/2012), the pdmH1N1 strain (A/California/04/2009), which served as the donor of the reassorted M gene, and other avian influenza strains (A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, and A/wild bird/South Korea/snu6/2018) exhibited marked resistance to amantadine even at the highest tested concentration of 1000 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings indicate that the acquisition of the M gene from pdmH1N1 conferred amantadine resistance to the reassorted cH3N2 virus.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Susceptibility of viruses to oseltamivir and zanamivir\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIt was found that the reassortment of the M gene did not affect the susceptibility of the virus to neuraminidase-targeting antiviral agents. Both the parental and M variant cH3N2 strains remained sensitive to oseltamivir, with IC\u003csub\u003e50\u003c/sub\u003e values of 0.002 \u0026micro;M and 0.005 \u0026micro;M, respectively. Similarly, both strains were sensitive to zanamivir, exhibiting an IC\u003csub\u003e50\u003c/sub\u003e values of 0.05 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggest that antiviral agents targeting proteins other than the M gene, such as neuraminidase inhibitors, retain efficacy regardless of M gene reassortment. Consistently, the pdmH1N1 and avian influenza strains also exhibited low IC\u003csub\u003e50\u003c/sub\u003e values against oseltamivir and zanamivir, confirming their maintained susceptibility to these drugs.\u003c/p\u003e\u003c/div\u003e\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\u003eHalf-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of influenza virus strains against amantadine\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVirus strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmantadine IC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmino Acid at Position 22\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eResponse to Amantadine\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/canine/Korea/01/2007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLeucine (L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSensitive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/canine/Korea/MV1/2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;1000\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eSerine (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/California/04/2009\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/chicken/Korea/ES/03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/aquatic bird/Korea/CN2/2009\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/wild bird/South Korea/snu6/2018\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\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe parental cH3N2 (A/canine/Korea/01/2007) strain exhibited an IC₅₀ of 0.1 \u0026micro;M against amantadine, indicating sensitivity. In contrast, the M variant cH3N2 (A/canine/Korea/MV1/2012) and other strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018) displayed drug resistance, as no measurable IC₅₀ to the amantadine could be determined.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHalf-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of influenza virus strains against oseltamivir and zanamivir\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVirus strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOseltamivir\u003c/p\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eResponse to\u003c/p\u003e\u003cp\u003eOseltamivir\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eZanamivir\u003c/p\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eResponse to\u003c/p\u003e\u003cp\u003eZanamivir\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/canine/Korea/01/2007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.002\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eSensitive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eSensitive\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/canine/Korea/MV1/2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.005\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/California/04/2009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.02\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/chicken/Korea/ES/03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.005\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/aquatic bird/Korea/CN2/2009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.05\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.2\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA/wild bird/South Korea/snu6/2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.001\u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026micro;M\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\u003eThe parental cH3N2 (A/canine/Korea/01/2007), the M variant cH3N2 (A/canine/Korea/MV1/2012), and other strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018) exhibited an IC₅₀ of 0.001\u0026ndash;0.05 \u0026micro;M against oseltamivir and 0.05\u0026ndash;0.2 \u0026micro;M against zanamivir, indicating moderate sensitivity to the neuraminidase-targeting antiviral agents.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Amino acid sequence comparison of the M2 protein\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo identify the molecular determinant responsible for amantadine resistance, the M2 protein sequences from the parental cH3N2, M variant cH3N2, and several amantadine-resistant strains were aligned and compared (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Multiple amino acid substitutions were observed across the M gene among these viruses. However, one specific position, amino acid residue 22, showed a unique pattern that strongly correlated with the antiviral phenotype.\u003c/p\u003e\u003cp\u003eAll amantadine-resistant viruses, including the M variant cH3N2, the pdmH1N1 strain, and the avian influenza strains, possessed a serine (S) residue at position 22, whereas the amantadine-sensitive parental cH3N2 uniquely retained a leucine (L) at this site. Although other substitutions were present, none showed a consistent difference between the resistant and sensitive groups. Therefore, residue 22 represents the only amino acid position that consistently distinguishes the amantadine-sensitive strain from all resistant strains, suggesting that the L22S substitution is the unique molecular determinant responsible for the observed amantadine resistance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis figure presents a comparison of amino acid sequences among various influenza virus strains, including parental cH3N2 (A/canine/Korea/01/2007), M variant cH3N2 (A/canine/Korea/MV1/2012), and other amantadine-resistant strains (A/California/04/2009, A/chicken/Korea/ES/03, A/aquatic bird/Korea/CN2/2009, A/wild bird/South Korea/snu6/2018). Notably, at position 22, only the parental cH3N2 strain possesses a leucine (L) residue, whereas all other strains have a serine (S) at this position. This unique substitution is exclusive to the parental cH3N2. These results suggest that the amino acid variation at position 22 may contribute to the distinct characteristics of the amantadine resistance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Predicted structural conformations and interactions of the M2 protein with amantadine\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo further investigate this hypothesis, we employed AlphaFold to predict the structural conformations of the M2 protein channels and their potential interactions with amantadine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Because neuraminidase-targeting antiviral agents do not interact with the M2 protein, interaction analysis was performed only between amantadine and the M2 protein of the parental cH3N2 and pdmH1N1 viruses. A structural comparison of the amino acid at position 22 revealed that in the parental cH3N2 virus, the leucine residue projects into the channel lumen, where it interacts with amantadine and contributes to the blockage of the channel. In contrast, in the resistant strains, the serine residue moves away from the channel lumen and is buried within the structure, resulting in an expanded channel that prevents amantadine from blocking the M2 ion channel, which is the mechanism of its antiviral activity.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Immunofluorescence assay (IFA)\u003c/h2\u003e\u003cp\u003eTo further assess the antiviral effects of amantadine, oseltamivir, and zanamivir, MDCK cells were infected with the same panel of influenza virus strains described above and treated with 100 \u0026micro;M of each compound for 12 h. Viral NP protein expression was examined by fluorescence microscope. In the cells infected with the viruses without drug treatment, strong NP fluorescence was observed, confirming successful viral infection. In contrast, NP expression was completely suppressed in parental cH3N2 infected cells treated with amantadine, with no detectable green fluorescence. However, cells infected with the M variant cH3N2, pdmH1N1, or avian influenza strains retained strong NP fluorescence despite amantadine treatment, indicating resistance to the drug. Treatment with oseltamivir or zanamivir resulted in a loss of NP fluorescence across all virus groups, consistent with inhibition of viral replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAlphaFold-predicted structures of the M2 ion channel from the parental cH3N2 virus (with leucine at position 22) and the human pandemic H1N1 virus (with serine at position 22) were shown. In the parental cH3N2 structure, the leucine residue (red sticks) at position 22 projects into the channel lumen, where it directly interacts with amantadine (orange cubic structure) and contributes to the effective blockage of the channel. In contrast, in the pandemic H1N1 structure, the serine residue (blue sticks) at the same position is oriented away from the channel lumen and is buried within the structure, resulting in an expanded channel. This conformational difference prevents amantadine from blocking the M2 ion channel in the pandemic H1N1 strain.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMCDK cells were infected with each influenza virus strain at 0.001 MOI, and viral NP protein expression was detected by IFA at 12 hours post-infection using an anti-NP antibody (green). The nucleus was counterstained with DAPI (blue). In cells infected with the parental cH3N2 strain and treated with amantadine, NP expression was completely suppressed, and no green fluorescence was detected. However, in the M variant cH3N2, pandemic H1N1, and avian influenza strains, strong cytoplasmic NP fluorescence persisted despite amantadine treatment, demonstrating resistance to the drug. Oseltamivir and zanamivir treatment led to marked reduction or complete loss of NP fluorescence across all virus groups, consistent with inhibition of viral replication.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.6. \u003cem\u003eIn vivo\u003c/em\u003e antiviral efficacy of amantadine\u003c/h2\u003e\u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e antiviral efficacy of amantadine, five-week-old female BALB/c mice were intranasally infected with either the parental or M variant cH3N2 virus, followed by oral administration of amantadine (100 mg/kg/day, twice daily) starting 4 h before infection and continuing every 12 h for 5 days. The virus control group, which served as the positive control, consisted of infected mice treated with PBS instead of amantadine. Lungs were collected at DPI 3 and 5. At DPI 3, amantadine treated group infected with the parental cH3N2 virus exhibited alleviated gross lung lesions characterized by lighter coloration and reduced consolidation compared with the virus control group. In contrast, lungs from the virus control group appeared dark red to brown, indicative of viral pneumonia and severe inflammation. However, no apparent differences were observed between amantadine treated and virus control groups in mice infected with the M variant cH3N2 virus, suggesting a loss of therapeutic efficacy in the M gene\u0026ndash;reassorted strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Consistent with these pathological findings, RT-qPCR analysis targeting the M gene revealed a significant reduction in viral RNA levels in amantadine treated mice infected with the parental cH3N2 strain. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). At DPI 3, viral RNA levels (expressed as 40 \u0026ndash; Cq values) were markedly lower in the treated group than in the untreated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and the difference remained significant at DPI 5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In contrast, no significant differences were observed between treated and untreated groups infected with the M variant cH3N2 strain (ns, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05 at both DPI 3 and 5). At DPI 3, amantadine treatment induced a significantly greater reduction of viral RNA in the parental cH3N2 group than in the M variant group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that the parental strain was more susceptible to the antiviral effect of amantadine. However, no significant difference between the two strains was observed at DPI 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThese results collectively demonstrate that amantadine effectively inhibits viral replication and mitigates lung pathology in mice infected with the parental cH3N2 virus, but fails to confer antiviral protection against the M gene-reassorted variant, highlighting the impact of M2 mutations on amantadine resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRepresentative lungs collected at DPI 3 from mice infected with either the parental or M variant cH3N2 virus. Amantadine treatment visibly reduced pulmonary lesions in parental cH3N2 infected mice, but not in M variant cH3N2 infected mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eViral RNA levels in lung homogenates were quantified by RT-qPCR targeting the M gene at DPI 3 and 5. (A and B) Amantadine treatment reduced viral RNA levels at DPI 3 and DPI 5 in parental cH3N2 infected mice, but not in M variant cH3N2 infected mice. Viral RNA reductions in the lungs of amantadine-treated mice were expressed as log₂-transformed values. (C) At DPI 3, amantadine treatment induced a significantly greater reduction of viral RNA in the parental cH3N2 group compared with the M variant group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas no significant difference was observed at DPI 5. Statistical analysis was performed using two-way ANOVA followed by Tukey\u0026rsquo;s multiple comparisons test. Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. ns, not significant; \u003cb\u003e*\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cb\u003e**\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003cb\u003e***\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we first demonstrated that canine influenza viruses acquiring the pdmH1N1-derived M segment through reassortment can subsequently showed resistance to amantadine. Furthermore, by comparing the amino acid sequences of amantadine-resistant and -sensitive strains, we identified the specific residues responsible for resistance acquisition. Structural modeling analysis further elucidated the underlying mechanism by which these mutations confer resistance. We figured out acquired amantadine resistance of reassortant canine influenza viruses carrying the pdmH1N1 M segment and proposed alternative antiviral drugs. In addition, by comparing the amino acid sequences of the M2 protein between amantadine-susceptible and -resistant strains followed by structural analysis, we identified a key substitution associated with resistance and demonstrated how this mutation affects drug efficacy.\u003c/p\u003e\u003cp\u003eGenetic reassortment, a common mechanism of influenza virus evolution, led to the emergence of a variant strain that exhibited resistance to amantadine. This phenotypic shift highlights the potential for rapid adaptation of influenza viruses under antiviral pressure, and underscores the importance of continuous molecular surveillance. Sequence alignment of the M2 protein across susceptible and resistant strains revealed a consistent amino acid substitution at position 22, where leucine (L) in the parental cH3N2 was replaced by serine (S) in resistant strains. Notably, this L22S mutation was the only consistent difference in M2 protein sequences correlating with the resistance phenotype, suggesting a causative role in mediating amantadine resistance.\u003c/p\u003e\u003cp\u003eStructural modeling using AlphaFold provided mechanistic insights into the functional impact of this substitution. In the susceptible virus, the leucine residue at position 22 protrudes into the M2 channel lumen, where it appears to directly participate in amantadine binding and the subsequent inhibition of ion conductance. In contrast, in the resistant strains, the serine residue is structurally repositioned away from the lumen and buried within the transmembrane domain, leading to a widened channel conformation that likely impairs amantadine binding and its inhibitory function. This structural alteration provides a plausible mechanistic explanation for the observed loss of drug efficacy.\u003c/p\u003e\u003cp\u003eWhile the current findings clearly demonstrate the structural and phenotypic basis of amantadine resistance, several experimental limitations should be acknowledged. The influenza virus strains used in this study exhibited relatively low pathogenicity in mice, preventing accurate assessment of survival rates following infection. Instead, viral replication in the lungs was quantified as a surrogate marker of \u003cem\u003ein vivo\u003c/em\u003e antiviral efficacy, which still provided a reliable measure of drug responsiveness. Moreover, reverse genetics-based point-mutation viruses were not generated to experimentally confirm the causal role of the identified M2 substitution. Nevertheless, the use of naturally occurring M variant isolated offers clinically meaningful evidence, as these field-derived viruses represent authentic resistance phenotypes circulating in nature.\u003c/p\u003e\u003cp\u003eDespite the acquired resistance to amantadine, the M variant cH3N2 retained susceptibility to neuraminidase (NA)-targeting antiviral agents, including oseltamivir and zanamivir. This observation is consistent with the conservation of the NA protein sequence and function, and suggests that these NA inhibitors remain viable treatment options in the context of M2-targeted drug resistance. These findings support the potential utility of combination antiviral therapy or NA inhibitor monotherapy as alternative strategies in cases where amantadine resistance is detected.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTaken together, this study not only identifies a specific amino acid mutation responsible for amantadine resistance in reassortant influenza viruses, but also demonstrates how structural modeling can complement sequence-based analyses to elucidate resistance mechanisms. Moreover, the maintenance of NA inhibitor susceptibility offers a practical therapeutic alternative and emphasizes the importance of multi-targeted antiviral development. Continued surveillance for resistance mutations and further functional validation of these findings \u003cem\u003ein vivo\u003c/em\u003e will be essential for guiding future antiviral strategies against evolving influenza viruses.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that resistance to amantadine can be acquired through reassortment with pandemic influenza viruses. Sequence and structural analyses identified a single M2 substitution, L22S, as the key determinant responsible for this resistance, providing a clear mechanistic explanation for impaired drug binding. Despite the acquisition of amantadine resistance, the retained susceptibility to NA inhibitors supports their continued usefulness as effective therapeutic options for controlling resistant variant strains. These finding highlight the evolutionary potential of influenza viruses to rapidly alter antiviral susceptibility and emphasize the need for ongoing molecular surveillance and diversified antiviral strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003epdmH1N1: 2009 pandemic H1N1 influenza virus\u003cbr\u003e\u0026nbsp;cH3N2: Canine H3N2 influenza virus\u003cbr\u003e\u0026nbsp;M1: Matrix protein 1\u003cbr\u003e\u0026nbsp;M2: Matrix protein 2\u003cbr\u003e\u0026nbsp;CPE: Cytopathic effect\u003cbr\u003eIC\u003csub\u003e50\u003c/sub\u003e: Half-maximal inhibitory concentration\u003cbr\u003e\u0026nbsp;MOI: Multiplicity of infection\u003cbr\u003eTCID\u003csub\u003e50\u003c/sub\u003e: 50% tissue culture infectious dose\u003cbr\u003e\u0026nbsp;IFA: Indirect immunofluorescence assay\u003cbr\u003e\u0026nbsp;NP: Nucleoprotein\u003cbr\u003e\u0026nbsp;NA: Neuraminidase\u003cbr\u003e\u0026nbsp;DPI: Day post infection\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (approval number SNU-250922-1). All procedures involving animals were conducted in accordance with the institutional guidelines for the care and use of laboratory animals and the national regulations of the Republic of Korea. Five-week-old female BALB/c mice (Koatech, Korea) were housed under specific-pathogen-free (SPF) conditions, and appropriate anesthesia and humane euthanasia procedures were employed to minimize animal suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe manuscript includes all datasets generated or analyzed during this study. Data will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through, High-Risk Animal infectious Disease Control Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (RS-2025-02304688) and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2024-00432287).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.G., E.B., X.X., and W.N. conceived and wrote the manuscript, E.G., X.X., M.Y., and D.S. contributed on \u003cem\u003ein vitro\u003c/em\u003e experiment, E.G.,and E.B. contributed on \u003cem\u003ein vivo\u003c/em\u003e experiment, J.H., J.-W.L. and W.N. made the figures, E.G., X.X., D.S., and W.N. reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEulhae Ga, Eunseo Bae and Xing xie contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Daesub Song ([email protected]) and Woonsung Na ([email protected])\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGaitonde DY, Moore FC, Morgan MK. Influenza: diagnosis and treatment. Am Family Phys. 2019;100(12):751\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong D, Kang B, Lee C, Jung K, Ha G, Kang D, et al. Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis. 2008;14(5):741.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong D, Moon H-J, An D-J, Jeoung H-Y, Kim H, Yeom M-J, et al. A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea. J Gen Virol. 2012;93(3):551\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoon H, Hong M, Kim J, Seon B, Na W, Park S-J, et al. H3N2 canine influenza virus with the matrix gene from the pandemic A/H1N1 virus: infection dynamics in dogs and ferrets. Epidemiol Infect. 2015;143(4):772\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBui M, Wills EG, Helenius A, Whittaker GR. Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol. 2000;74(4):1781\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePielak RM, Chou JJ. Influenza M2 proton channels. Biochim et Biophys Acta (BBA)-Biomembranes. 2011;1808(2):522\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHay A, Wolstenholme A, Skehel J, Smith MH. The molecular basis of the specific anti-influenza action of amantadine. EMBO J. 1985;4(11):3021\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCho H-G, Choi J-H, Kim W-H, Hong H-K, Yoon M-H, Jho E-H, et al. High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005\u0026ndash;2010. Arch Virol. 2013;158:241\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaraket H, Saito R, Suzuki Y, Baranovich T, Dapat C, Caperig-Dapat I, et al. Genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza A/H1N1 viruses. J Clin Microbiol. 2010;48(4):1085\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong F, Krishnan A, Zheng EJ, St\u0026auml;rk H, Manson AL, Earl AM, et al. Benchmarking AlphaFold-enabled molecular docking predictions for antibiotic discovery. Mol Syst Biol. 2022;18(9):e11081.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGutnik D, Evseev P, Miroshnikov K, Shneider M. Using AlphaFold predictions in viral research. Curr Issues Mol Biol. 2023;45(4):3705\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesai D, Kantliwala SV, Vybhavi J, Ravi R, Patel H, Patel J. Review of AlphaFold 3: transformative advances in drug design and therapeutics. Cureus. 2024;16(7):e63646.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHoffmann E, Stech J, Guan Y, Webster R, Perez D. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol. 2001;146:2275\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen Y, Cui D, Zheng S, Yang S, Tong J, Yang D, et al. Simultaneous detection of influenza A, influenza B, and respiratory syncytial viruses and subtyping of influenza A H3N2 virus and H1N1 (2009) virus by multiplex real-time PCR. J Clin Microbiol. 2011;49(4):1653\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNelson MI, Vincent AL. Reverse zoonosis of influenza to swine: new perspectives on the human\u0026ndash;animal interface. Trends Microbiol. 2015;23(3):142\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Influenza A virus, reassortment, amantadine, antiviral drug, resistance","lastPublishedDoi":"10.21203/rs.3.rs-8173350/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8173350/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel variant of the canine influenza virus H3N2 (cH3N2), designated as the M variant, was identified to contain a matrix (M) gene segment derived from the 2009 pandemic H1N1 virus (pdmH1N1), raising concerns regarding potential changes in antiviral drug sensitivity. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antiviral susceptibility assays demonstrated that while the parental cH3N2 strain was sensitive to amantadine, the M variant had acquired resistance to this drug. In contrast, both strains remained susceptible to neuraminidase inhibitors such as oseltamivir and zanamivir. Comparative amino acid sequence analysis of the M2 protein identified a substitution, L22S, uniquely present in amantadine resistant strains. Structural modeling of the M2 ion channel also suggested that the amantadine resistance observed in the M variant results from conformational alterations that impede drug binding. Collectively, these findings indicate that genetic reassortment with pdmH1N1 confers amantadine resistance in cH3N2 through the L22S substitution in the M2 protein. The preserved susceptibility to neuraminidase inhibitors suggests that these agents remain effective alternatives for controlling resistant strains, emphasizing the importance of continued molecular surveillance and diversified antiviral strategies.\u003c/p\u003e","manuscriptTitle":"Acquisition of Amantadine Resistance via M Gene Reassortment in Canine H3N2 Influenza Virus and Elucidation of the Resistance Mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 15:30:07","doi":"10.21203/rs.3.rs-8173350/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-09T16:38:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T16:20:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T15:57:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118849422305561572080692646396844166567","date":"2025-11-27T15:42:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298039167505831855750402003694509092738","date":"2025-11-27T13:44:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-25T10:10:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-24T10:06:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-24T07:59:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2025-11-21T11:42:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e36e9865-579c-4edc-8ebe-56eb44e32a81","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-8173350","link":"https://doi.org/10.1186/s12985-026-03097-2","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2026-02-13 15:58:17","publishedOnDateReadable":"February 13th, 2026"},"versionCreatedAt":"2025-11-28 15:30:07","video":"","vorDoi":"10.1186/s12985-026-03097-2","vorDoiUrl":"https://doi.org/10.1186/s12985-026-03097-2","workflowStages":[]},"version":"v1","identity":"rs-8173350","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8173350","identity":"rs-8173350","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}