Astragaloside IV inhibits inflammation caused by influenza virus via reactive oxygen species/NOD-like receptor thermal protein domain associated protein 3/Caspase-1 signaling pathway.

BACKGROUND: Astragaloside IV (AS-IV) is the most active monomer in the traditional Chinese herbal medicine Radix Astragali, which has a wide range of antiviral, anti-inflammatory, and antifibrosis pharmacological effects, and shows protective effects in acute lung injury. METHODS: This study utilized the immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, and hematoxylin and eosin staining methods to investigate the mechanism of AS-IV in reducing viral pneumonia caused by influenza A virus in A549 cells and BALB/c mice. RESULTS: The results showed that AS-IV suppressed reactive oxygen species production in influenza virus-infected A549 cells in a dose-dependent manner, and subsequently inhibited the activation of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 inflammasome and Caspase-1, decreased interleukin (IL) -1ß and IL-18 secretion. In BALB/c mice infected with Poly (I:C), oral administration of AS-IV can significantly reduce Poly (I:C)-induced acute pneumonia and lung pathological injury. CONCLUSIONS: AS-IV alleviates the inflammatory response induced by influenza virus in vitro and lung flammation and structural damage caused by poly (I:C) in vivo.

Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

Molecular screening of phytocompounds targeting the interface between influenza A NS1 and TRIM25 to enhance host immune responses.

BACKGROUND: Influenza A virus causes severe respiratory illnesses, especially in developing nations where most child deaths under 5 occur due to lower respiratory tract infections. The RIG-I protein acts as a sensor for viral dsRNA, triggering interferon production through K63-linked poly-ubiquitin chains synthesized by TRIM25. However, the influenza A virus's NS1 protein hinders this process by binding to TRIM25, disrupting its association with RIG-I and preventing downstream interferon signalling, contributing to the virus's evasion of the immune response. METHODS: In our study we used structural-based drug designing, molecular simulation, and binding free energy approaches to identify the potent phytocompounds from various natural product databases (>100,000 compounds) able to inhibit the binding of NS1 with the TRIM25. RESULTS: The molecular screening identified EA-8411902 and EA-19951545 from East African Natural Products Database, NA-390261 and NA-71 from North African Natural Products Database, SA-65230 and SA- 4477104 from South African Natural Compounds Database, NEA- 361 and NEA- 4524784 from North-East African Natural Products Database, TCM-4444713 and TCM-6056 from Traditional Chinese Medicines Database as top hits. The molecular docking and binding free energies results revealed that these compounds have high affinity with the specific active site residues (Leu95, Ser99, and Tyr89) involved in the interaction with TRIM25. Additionally, analysis of structural dynamics, binding free energy, and dissociation constants demonstrates a notably stronger binding affinity of these compounds with the NS1 protein. Moreover, all selected compounds exhibit exceptional ADMET properties, including high water solubility, gastrointestinal absorption, and an absence of hepatotoxicity, while adhering to Lipinski's rule. CONCLUSION: Our molecular simulation findings highlight that the identified compounds demonstrate high affinity for specific active site residues involved in the NS1-TRIM25 interaction, exhibit exceptional ADMET properties, and adhere to drug-likeness criteria, thus presenting promising candidates for further development as antiviral agents against influenza A virus infections.

Low antiviral resistance in Influenza A and B viruses isolated in Mexico from 2010 to 2023.

The most widely used class of antivirals available for Influenza treatment are the neuraminidase inhibitors (NAI) Oseltamivir and Zanamivir. However, amino acid (AA) substitutions in the neuraminidase may cause reduced inhibition or high antiviral resistance. In Mexico, the current state of knowledge about NAI susceptibility is scarce, in this study we report the results of 14 years of Influenza surveillance by phenotypic and genotypic methods. A total of 255 isolates were assessed with the NAI assay, including Influenza A(H1N1)pdm09, A(H3N2) and Influenza B (IBV). Furthermore, 827 sequences contained in the GISAID platform were analyzed in search of relevant mutations.Overall, five isolates showed highly reduced inhibition or reduced inhibition to Oseltamivir, and two showed reduced inhibition to Zanamivir in the NAI assays. Additionally, five A(H1N1)pdm09 sequences from the GISAID possessed AA substitutions associated to reduced inhibition to Oseltamivir and none to Zanamivir. Oseltamivir resistant A(H1N1)pdm09 harbored the H275Y mutation. No genetic mutations were identified in Influenza A(H3N2) and IBV. Overall, these results show that in Mexico the rate of NAI resistance is low (0.6%), but it is essential to continue the Influenza surveillance in order to understand the drug susceptibility of circulating strains.

Effect of Haoqin Qingdan Tang on influenza A virus through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway.

OBJECTIVE: Influenza, a viral respiratory illness, leads to seasonal epidemics and occasional pandemics. Given the rising resistance and adverse reactions associated with anti-influenza drugs, Traditional Chinese Medicine (TCM) emerges as a promising approach to counteract the influenza virus. Specifically, Haoqin Qingdan Tang (HQQDT), a TCM formula, has been employed as an adjuvant treatment for influenza in China. However, the active compounds and underlying mechanisms of HQQDT remain unknown. AIM: The aim of this study was to investigate HQQDT's antiviral and anti-inflammatory activities in both in vivo and in vitro, and further reveal its active ingredients and mechanism. METHODS: In vivo and in vitro experiments were conducted to verify the antiviral and anti-inflammatory activities of HQQDT. Subsequently, the active ingredients and mechanism of HQQDT were explored through combining high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS) analysis and network pharmacology. Finally, the examinations of cell cytokines and signaling pathways aimed to elucidate the predicted mechanisms. RESULTS: The results indicated that HQQDT exhibited inhibitory effects on influenza viruses A/PR/8/34 (H1N1), A/HK/1/68 (H3N2), and A/California/4/2009 (H1N1) in vitro. Furthermore, HQQDT enhanced the survival rate of influenza-infected mice, reduced the lung index and lung virus titer, and mitigated lung tissue damage in vivo. The proinflammatory cytokine expression levels upon influenza virus infection in PR8-induced A549 cells or mice were suppressed by HQQDT, including IL-6, IL-1ß, CCL2, CCL4, IP-10, interferon ß1 (IFN-ß1), the interferon regulatory factor 3 (IRF3), and hemagglutinin (HA). Twenty-two active components of HQQDT against influenza were identified using HPLC-Q-TOF-MS analysis. Based on network pharmacological predictions, the JAK/STAT signaling pathway is considered the most relevant for HQQDT's action against influenza. Finally, western blot assays revealed that HQQDT regulated the protein level of the JAK/STAT signaling pathway in PR8-infected A549 cells and lung tissue. CONCLUSION: These findings verified the antiviral and anti-inflammatory effects of HQQDT through JAK-STAT signaling pathway in influenza infections, laying the foundation for its further development.

Exploring the Antiviral Potential of Natural Compounds against Influenza: A Combined Computational and Experimental Approach.

The influenza A virus nonstructural protein 1 (NS1), which is crucial for viral replication and immune evasion, has been identified as a significant drug target with substantial potential to contribute to the fight against influenza. The emergence of drug-resistant influenza A virus strains highlights the urgent need for novel therapeutics. This study proposes a combined theoretical criterion for the virtual screening of molecular libraries to identify candidate NS1 inhibitors. By applying the criterion to the ZINC Natural Product database, followed by ligand-based virtual screening and molecular docking, we proposed the most promising candidate as a potential NS1 inhibitor. Subsequently, the selected natural compound was experimentally evaluated, revealing measurable virus replication inhibition activity in cell culture. This approach offers a promising avenue for developing novel anti-influenza agents targeting the NS1 protein.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Production of Disinfective Coating Layer to Facial Masks Supplemented with Camellia sinensis Extract.

Although usefulness of masks for protection against respiratory pathogens, accumulation of pathogens on their surface represents a source of infection spread. Here we prepared a plant extract-based disinfecting layer to be used in coating masks thus inhibiting their capacity to transmit airborne pathogens. To reach this, a polypropylene membrane base was coated with a layer of polyvinyledine difluoride polymer containing 500 µg/ml of Camellia sinensis (Black tea) methanolic extract. Direct inhibitory effects of C. sinensis were initially demonstrated against Staphylococcus aureus (respiratory bacteria), influenza A virus (enveloped virus) and adenovirus 1 (non-enveloped virus) which were directly proportional to both extract concentration and incubation time with the pathogen. This was later confirmed by the capacity of the supplemented membrane with the plant extract to block infectivity of the above mentioned pathogens, recorded % inhibition values were 61, 72 and 50 for S. aureus, influenza and adenovirus, respectively. In addition to the disinfecting capacity of the membrane its hydrophobic nature and pore size (154 nm) prevented penetration of dust particles or water droplets carrying respiratory pathogens. In summary, introducing this layer could protect users from infection and decrease infection risk upon handling contaminated masks surfaces.

Spleen tyrosine kinase inhibitor R406 has both antiviral and anti-inflammatory effects on severe influenza A infection.

Death due to severe influenza is usually a fatal complication of a dysregulated immune response more than the acute virulence of an infectious agent. Although spleen tyrosine kinase (SYK) as a critical immune signaling molecule and therapeutic target plays roles in airway inflammation and acute lung injury, the role of SYK in influenza virus infection is not clear. Here, we investigated the antiviral and anti-inflammatory effects of SYK inhibitor R406 on influenza infection through a coculture model of human alveolar epithelial (A549) and macrophage (THP-1) cell lines and mouse model. The results showed that R406 treatment increased the viability of A549 and decreased the pathogenicity and mortality of lethal influenza virus in mice with influenza A infection, decreased levels of intracellular signaling molecules under the condition of inflammation during influenza virus infection. Combination therapy with oseltamivir further ameliorated histopathological damage in the lungs of mice and further delayed the initial time to death compared with R406 treatment alone. This study demonstrated that phosphorylation of SYK is involved in the pathogenesis of influenza, and R406 has antiviral and anti-inflammatory effects on the treatment of the disease, which may be realized through multiple pathways, including the already reported SYK/STAT/IFNs-mediated antiviral pathway, as well as TNF-α/SYK- and SYK/Akt-based immunomodulation pathway.

An oleanic acid decorated gold nanorod for highly efficient inhibition of hemagglutinin and visible rapid detection of the influenza virus.

Accurate diagnosis and effective antiviral treatments are urgently needed for the prevention and control of flu caused by influenza viruses. In this study, a novel oleanic acid (OA) functionalized gold nanorod OA-AuNP was prepared through a convenient ligand-exchange reaction. As hemagglutinin (HA) on the viral surface binds strongly to the multiple OA molecules on the surface of the nanoparticle, the prepared OA-AuNP was found to exhibit potent antiviral activity against a wide range of influenza A virus strains. Furthermore, the change in color resulting from the specific binding between HA and OA and the resultant aggregation of the OA-AuNP can be visually observed or measured by UV-vis spectra with a detection limit of 2 and 0.18 hemagglutination units (HAU), respectively, which is comparable to the commercially available influenza colloid gold rapid diagnostic kits. These findings demonstrate the potential of the OA-AuNP for the development of novel multivalent antiviral conjugates and the diagnosis of influenza virus.

Inhibition Effects and Mechanisms of Marine Compound Mycophenolic Acid Methyl Ester against Influenza A Virus.

Influenza A virus (IAV) can cause infection and illness in a wide range of animals, including humans, poultry, and swine, and cause annual epidemics, resulting in thousands of deaths and millions of hospitalizations all over the world. Thus, there is an urgent need to develop novel anti-IAV drugs with high efficiency and low toxicity. In this study, the anti-IAV activity of a marine-derived compound mycophenolic acid methyl ester (MAE) was intensively investigated both in vitro and in vivo. The results showed that MAE inhibited the replication of different influenza A virus strains in vitro with low cytotoxicity. MAE can mainly block some steps of IAV infection post adsorption. MAE may also inhibit viral replication through activating the cellular Akt-mTOR-S6K pathway. Importantly, oral treatment of MAE can significantly ameliorate pneumonia symptoms and reduce pulmonary viral titers, as well as improving the survival rate of mice, and this was superior to the effect of oseltamivir. In summary, the marine compound MAE possesses anti-IAV effects both in vitro and in vivo, which merits further studies for its development into a novel anti-IAV drug in the future.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Curcumin as a natural potential drug candidate against important zoonotic viruses and prions: A narrative review.

Zoonotic diseases are major public health concerns and undeniable threats to human health. Among Zoonotic diseases, zoonotic viruses and prions are much more difficult to eradicate, as they result in higher infections and mortality rates. Several investigations have shown curcumin, the active ingredient of turmeric, to have wide spectrum properties such as anti-microbial, anti-vascular, anti-inflammatory, anti-tumor, anti-neoplastic, anti-oxidant, and immune system modulator properties. In the present study, we performed a comprehensive review of existing in silico, in vitro, and in vivo evidence on the antiviral (54 important zoonotic viruses) and anti-prion properties of curcumin and curcuminoids in PubMed, Google Scholar, Science Direct, Scopus, and Web of Science databases. Database searches yielded 13,380 results, out of which 216 studies were eligible according to inclusion criteria. Of 216 studies, 135 (62.5%), 24 (11.1%), and 19 (8.8%) were conducted on the effect of curcumin and curcuminoids against SARS-CoV-2, Influenza A virus, and dengue virus, respectively. This review suggests curcumin and curcuminoids as promising therapeutic agents against a wide range of viral zoonoses by targeting different proteins and signaling pathways.

Inhibition of influenza A virus and SARS-CoV-2 infection or co-infection by griffithsin and griffithsin-based bivalent entry inhibitor.

Outbreaks of acute respiratory viral diseases, such as influenza and COVID-19 caused by influenza A virus (IAV) and SARS-CoV-2, pose a serious threat to global public health, economic security, and social stability. This calls for the development of broad-spectrum antivirals to prevent or treat infection or co-infection of IAV and SARS-CoV-2. Hemagglutinin (HA) on IAV and spike (S) protein on SARS-CoV-2, which contain various types of glycans, play crucial roles in mediating viral entry into host cells. Therefore, they are key targets for the development of carbohydrate-binding protein-based antivirals. This study demonstrated that griffithsin (GRFT) and the GRFT-based bivalent entry inhibitor GL25E (GRFT-L25-EK1) showed broad-spectrum antiviral effects against IAV infection in vitro by binding to HA in a carbohydrate-dependent manner and effectively protected mice from lethal IAV infection. Although both GRFT and GL25E could inhibit infection of SARS-CoV-2 Omicron variants, GL25E proved to be significantly more effective than GRFT and EK1 alone. Furthermore, GL25E effectively inhibited in vitro co-infection of IAV and SARS-CoV-2 and demonstrated good druggability, including favorable safety and stability profiles. These findings suggest that GL25E is a promising candidate for further development as a broad-spectrum antiviral drug for the prevention and treatment of infection or co-infection from IAV and SARS-CoV-2.IMPORTANCEInfluenza and COVID-19 are highly contagious respiratory illnesses caused by the influenza A virus (IAV) and SARS-CoV-2, respectively. IAV and SARS-CoV-2 co-infection exacerbates damage to lung tissue and leads to more severe clinical symptoms, thus calling for the development of broad-spectrum antivirals for combating IAV and SARS-CoV-2 infection or co-infection. Here we found that griffithsin (GRFT), a carbohydrate-binding protein, and GL25E, a recombinant protein consisting of GRFT, a 25 amino acid linker, and EK1, a broad-spectrum coronavirus inhibitor, could effectively inhibit IAV and SARS-CoV-2 infection and co-infection by targeting glycans on HA of IAV and spike (S) protein of SARS-CoV-2. GL25E is more effective than GRFT because GL25E can also interact with the HR1 domain in SARS-CoV-2 S protein. Furthermore, GL25E possesses favorable safety and stability profiles, suggesting that it is a promising candidate for development as a drug to prevent and treat IAV and SARS-CoV-2 infection or co-infection.

Influenza C virus susceptibility to antivirals with different mechanisms of action.

Antiviral susceptibility of influenza viruses was assessed using a high-content imaging-based neutralization test. Cap-dependent endonuclease inhibitors, baloxavir and AV5116, were superior to AV5115 against type A viruses, and AV5116 was most effective against PA mutants tested. However, these three inhibitors displayed comparable activity (EC50 8-22 nM) against type C viruses from six lineages. Banana lectin and a monoclonal antibody, YA3, targeting the hemagglutinin-esterase protein effectively neutralized some, but not all, type C viruses.

Novel Anti-Viral Properties of the Herbal Extract of Davallia mariesii against Influenza A Virus.

Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.

Intranasal neomycin evokes broad-spectrum antiviral immunity in the upper respiratory tract.

Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.

Compounds based on Adamantyl-substituted Amino Acids and Peptides as Potential Antiviral Drugs Acting as Viroporin Inhibitors.

The discussion has revolved around the derivatives of amino acids and peptides containing carbocycles and their potential antiviral activity in vitro against influenza A, hepatitis C viruses, and coronavirus. Studies conducted on cell cultures reveal that aminoadamantane amino acid derivatives exhibit the capacity to hinder the replication of viruses containing viroporins. Furthermore, certain compounds demonstrate potent virucidal activity with respect to influenza A/H5N1 and hepatitis C virus particles. A conceptual framework for viroporin inhibitors has been introduced, incorporating carbocyclic motifs as membranotropic carriers in the structure, alongside a functional segment comprised of amino acids and peptides. These components correspond to the interaction with the inner surface of the channel's pore or another target protein.

Morpholinodiazenyl chalcone blocks influenza A virus capsid uncoating by perturbing the clathrin-mediated vesicular trafficking pathway.

Influenza A virus (IAV) is a highly contagious respiratory pathogen that significantly threatens global health by causing seasonal epidemics and occasional, unpredictable pandemics. To identify new compounds with therapeutic potential against IAV, we designed and synthesized a series of 4'-morpholinodiazenyl chalcones using the molecular hybridization method, performed a high-content screen against IAV, and found that (E)-1-{4-[(E)-morpholinodiazenyl]phenyl}-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (MC-22) completely neutralized IAV infection. While MC-22 allowed IAV to successfully internalize into the cell and fuse at the acidic late endosomes, it prevented viral capsid uncoating and genome release. Since IAV majorly utilizes clathrin-mediated endocytosis (CME) for cellular entry, we examined whether MC-22 had any effect on CME, using nonviral cargoes that enter cells via clathrin-dependent or -independent pathways. Although MC-22 showed no effect on the uptake of choleratoxin B, a cargo that enters cells majorly via the clathrin-independent pathway, it significantly attenuated the clathrin-dependent internalization of both epidermal growth factor and transferrin. Cell biological analyses revealed a marked increase in the size of early endosomes upon MC-22 treatment, indicating an endosomal trafficking/maturation defect. This study reports the identification of MC-22 as a novel CME-targeting, highly potent IAV entry inhibitor, which is expected to neutralize a broad spectrum of viruses that enter the host cells via CME.