Development Of Anti-influenza Drugs Research Articles

HACD3 Prevents PB1 from Autophagic Degradation to Facilitate the Replication of Influenza A Virus.

Influenza A virus (IAV) continues to pose serious threats to the global animal industry and public health security. Identification of critical host factors engaged in the life cycle of IAV and elucidation of the underlying mechanisms of their action are particularly important for the discovery of potential new targets for the development of anti-influenza drugs. Herein, we identified Hydroxyacyl-CoA Dehydratase 3 (HACD3) as a new host factor that supports the replication of IAV. Downregulating the expression of HACD3 reduced the level of viral PB1 protein in IAV-infected cells and in cells that were transiently transfected to express PB1. Silencing HACD3 expression had no effect on the level of PB1 mRNA but could promote the lysosome-mediated autophagic degradation of PB1 protein. Further investigation revealed that HACD3 interacted with PB1 and selective autophagic receptor SQSTM1/p62, and HACD3 competed with SQSTM1/p62 for the interaction with PB1, which prevented PB1 from SQSTM1/p62-mediated autophagic degradation. Collectively, these findings establish that HACD3 plays a positive regulatory role in IAV replication by stabilizing the viral PB1 protein.

Discovery and synthesis of novel benzoylhydrazone neuraminidase inhibitors

Neuraminidase (NA) serves as a promising target for the exploration and development of anti-influenza drugs. In this work, lead compound 5 was discovered through pharmacophore-based virtual screening and molecular dynamics simulation, and 14 new compounds were obtained by modifying the lead compound 5 based on pharmacophore features. The biological activity test shows that 5n (IC50 = 0.13 μM) has a better inhibitory effect on wild-type NA (H5N1), while 5i (IC50 = 0.44 μM) has a prominent inhibitory effect on mutant NA (H5N1-H274Y), both of them are better than the positive control oseltamivir carboxylate (OSC). The analysis of docking results indicate that the good activities of compounds 5n and 5i may be attributed to the thiophene ring in 5n can stretch into the 150-cavity of NA, whereas the thiophene moiety in 5i can extend to the 430-cavity of NA. The findings of this study may be helpful for the discovery of new NA inhibitors.

Progress of Influenza Viruses and Inhibitors.

Influenza is an acute respiratory disease caused by influenza viruses. It has the characteristics of fast transmission and strong infectivity, and it does great harm to human health and survival. It is estimated that the seasonal influenza epidemics every year will cause about one billion cases of infections and hundreds of thousands of deaths worldwide, while influenza A virus is the leading cause of infection and death. Currently, the main drugs used in clinics to treat influenza viruses are neuraminidase inhibitors, and these drugs have shown excellent efficacy in treating influenza viruses. However, various mutant strains have developed resistance to these effective drugs in clinics (such as the subtype mutant strains of H274Y in H1N1 or H5N1 and E119V in H3N2 have developed resistance to Oseltamivir). Influenza viruses mutate frequently, and new viral strains are constantly discovered, and the pandemics will break out at any time. Therefore, it is urgent to develop efficient and broad-spectrum drugs to prevent and treat the influenza pandemic caused by the emerging new subtypes. This review focuses on describing the pandemic history, the structure, function and prevention methods of influenza viruses and the progress of the development of anti-influenza drugs, to provide the reference for prevention and treatment of influenza viruses and development of influenza virus inhibitors.

Genome-wide CRISPR screen identifies GNE as a key host factor that promotes influenza A virus adsorption and endocytosis

ABSTRACT Replication of influenza A virus (IAV) is highly reliant on host cell function, and to identify the key host factor required in the influenza A virus life cycle, a genome-wide CRISPR/Cas9 knockout (KO) screen was conducted in A549 cells infected by H1N1 influenza virus. The results showed that glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) plays a crucial role in the replication of influenza A virus, and knockout of the GNE significantly reduced the replication of multiple subtype influenza A viruses in A549 cells, and restoration of the GNE expression in the GNE-knockout cells partially recovered IAV infection. Additionally, overexpression of the GNE in wild-type (WT) cells promoted IAV infection to a certain extent. Further study showed that the GNE is involved in α-2,3- and α-2,6-linked sialic acid (Sia) synthesis, which is the major receptor of the influenza A virus, and the GNE knockout downregulated the α-2,3- and α-2,6-linked sialic acid expression. In summary, the knockout of the GNE inhibits adsorption and endocytosis of IAV. This study provides a new approach to elucidate the replication mechanism of IAV and identifies GNE as a potential target for anti-influenza drug development. IMPORTANCE Influenza A virus infection requires the assistance of the host proteins. Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) was identified as an important host factor in influenza A virus infection by genome-wide CRISPR screening. GNE knockout (KO) extensively inhibited the replication of multiple subtype influenza A viruses. It indirectly participated in IAV adsorption and endocytosis by regulating the expression of sialic acid (Sia), the main receptor of IAV. This finding provides novel insights into the replication mechanism of IAV.

Antiviral effect of the Ferula Kuhistanica Korovin plant, growing in the high-mountain conditions of the Republic of Tajikistan

This work presents the results of studies on the extract obtained from the root of Ferula kuhistanica Korov, in relation to 2 strains of the influenza virus: A/Vlad/2/09(H1N1) and A/Almaty/8/98(H3N2). It has been shown that the F. kuhistanica Korov extract exhibits selective antiviral activity and primarily acts on the influenza virus strain A/Vlad/2/09(H1N1), not showing an inhibitory effect against another antigenic variant of this virus – the strain A/ Almaty/8/98(H3N2). The tested extract was characterized by high IC50, EC50, and IS50 indicators. Its chemotherapeutic effect (XTI) is 23 times higher than that of the commercial anti-influenza drug - Tamiflu. The data obtained open up prospects for the possible use of extracts obtained from the root of various species of the genus Ferula, growing in our country, in the development of anti-influenza drugs.

Phenolic compound SG-1 from Balanophora harlandii and its derivatives exert anti-influenza A virus activity via activation of the Nrf2/HO-1 pathway

Influenza A virus (IAV) is one of the leading causes of respiratory illness and continues to cause pandemics around the world. Against this backdrop, drug resistance poses a challenge to existing antiviral drugs, and hence, there is an urgent need for developing new antiviral drugs. In this study, we obtained a phenolic compound SG-7, a derivative of natural compound 2-hydroxymethyl-1,4-hydroquinone, which exhibits inhibitory activity toward a panel of influenza viruses and has low cellular toxicity. Mechanistic studies have shown that SG-7 exerts its anti-IAV properties by acting on the virus itself and modulating host signaling pathways. Namely, SG-7 targets the HA2 subunit of hemagglutinin (HA) to block the fusion of viral-cellular membranes and inhibits IAV-induced oxidative stress and overexpression of pro-inflammatory factors by activating the Nrf2/HO-1 pathway and reducing NF-κB activation. In addition, SG-7 can enhance type I IFN antiviral response by inducing Nrf2 expression. Importantly, SG-7 showed the ability to inhibit viral replication in the lungs of IAV-infected mice and reduce their mortality. Therefore, SG-7 may be a promising lead compound for anti-influenza drug development.

Interaction of Influenza A Nucleoprotein with Host hnRNP-C Is Implicated in Viral Replication.

The host interactome of influenza viral proteins is ever-expanding. In this work, we report the identification of host heterogeneous nuclear ribonucleoprotein C (hnRNP-C) as an interacting partner of influenza A virus nucleoprotein (NP). We confirmed that this interaction exists across different influenza A subtypes and strains. Using biochemical methods, we determined that hnRNP-C interacts with NP via its C-terminal auxiliary domain. Further, we determined that the hnRNP-C is a negative regulator of influenza viral growth. Its interaction with NP is implicated in the promotion of host cell apoptosis during viral infection. It is the first time that the interaction between influenza nucleoprotein and host heterogeneous nuclear ribonucleoprotein C is characterized in detail. Overall, these findings not only characterize the interaction between NP and its host interacting partner hnRNP-C but also clarify the functional significance of this interaction. This work may lead to a new therapeutic target for the development of anti-influenza drugs.

A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

ABSTRACT The quadrilateral reassortant IAV A/(H1N1) pdm09 is the pathogen responsible for the first influenza pandemic of the 21st century. The virus spread rapidly among hosts causing high mortality within human population. Efficient accumulation of virions is known to be important for the rapid transmission of virus. However, the mechanism by which A/(H1N1) pdm09 promotes its rapid replication has not been fully studied. Here, we found the NS1 of A/(H1N1) pdm09 mediated complete macroautophagy/autophagy, and then facilitated self-replication, which may be associated with the more rapid spread of this virus compared with H1N1WSN and H3N8JL89. We found that the promotion of self-replication could be mainly attributed to NS1pdm09 strongly antagonizing the inhibitory effect of LRPPRC on autophagy. The interaction between NS1pdm09 and LRPPRC competitively blocked the interaction of LRPPRC with BECN1/Beclin1, resulting in increased recruitment of BECN1 for PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and induction of the initiation of autophagy. In conclusion, we uncover the unique molecular mechanism by which A/(H1N1) pdm09 utilizes autophagy to promote self-replication, and we provide theoretical basics for the analysis of the etiological characteristics of the A/(H1N1) pdm09 pandemic and the development of anti-influenza drugs and vaccines. Abbreviations: 293T: human embryonic kidney 293 cells; 293T_LRPPRC: stable LRPPRC expression 293T cells; 3-MA: 3-methyladenine; A549 cells: human non-small cell lung cancer cells; AA: amino acid; ACTB: actin beta; BECN1: beclin 1; BECN1 KO: BECN1 knockout 293T cells; Cal: calyculin A; Co-IP: co-immunoprecipitation; CQ: chloroquine; DC: dendritic cell; Eug: eugenol; GFP: green fluorescent protein; HA: hemagglutinin; HIV: human immunodeficiency virus; IAVs: Influenza A viruses; IFN: interferon; JL89: A/equine/Jilin/1/1989 (H3N8); LAMP2: lysosomal associated membrane protein 2; LRPPRC: leucine rich pentatriicopeptide repeat containing; LRPPRC KO: LRPPRC knockout 293T cells; M2: matrix 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MDCK: Madin-Darby canine kidney cells; MOI: multiplicity of infection; MS: mass spectrometry; NP: nucleoprotein; NS1: non-structural protein 1; NS1JL89: non-structural protein 1 of A/equine/Jilin/1/1989 (H3N8); NS1pdm09: non-structural protein 1 of A/(H1N1) pdm09; NS1SC09: non-structural protein 1 of A/Sichuan/2009 (H1N1); NS1WSN: non-structural protein 1 of A/WSN/1933 (H1N1); PB1: polymerase basic protein 1; PB1-F2: alternate reading frame discovered in PB1 gene segment; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PR8: A/PR/8/34 (H1N1); Rapa: rapamycin; RFP: red fluorescent protein; SC09: A/Sichuan/2009 (H1N1); SQSTM1/p62: sequestosome 1; STK4/MST1: serine/threonine kinase 4; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; WHO: World Health Organization; WSN: A/WSN/1933 (H1N1); WSN-NS1JL89: WSN recombinant strain in which NS1 was replaced with that of JL89; WSN-NS1SC09: WSN recombinant strain in which NS1 was replaced with that of SC09.

Identification of cytochrome c oxidase subunit 4 isoform 1 as a positive regulator of influenza virus replication.

Human infection with highly pathogenic H5N1 influenza virus causes severe respiratory diseases. Currently, the drugs against H5N1 are limited to virus-targeted inhibitors. However, drug resistance caused by these inhibitors is becoming a serious threat to global public health. An alternative strategy to reduce the resistance risk is to develop antiviral drugs targeting host cell proteins. In this study, we demonstrated that cytochrome c oxidase subunit 4 isoform 1 (COX41) of host cell plays an important role in H5N1 infection. Overexpression of COX41 promoted viral replication, which was inhibited by silencing or knockout the expression of COX41 in the host cell. The ribonucleoproteins (RNPs) of H5N1 were retained in the cell nucleus after knockout cellular COX41. Strikingly, inhibition of cellular COX41 by lycorine, a small-molecule compound isolated from Amaryllidaceae plants, reduced the levels of COX41-induced ROS and phosphorylation of extracellular signal-regulated kinase (ERK) in cells, thus resulting in the blockage of nuclear export of vRNP and inhibition of viral replication. In H5N1-infected mice that were treated with lycorine, we observed a reduction of viral titers and inhibition of pathological changes in the lung and trachea tissues. Importantly, no resistant virus was generated after culturing the virus with the continuous treatment of lycorine. Collectively, these findings suggest that COX41 is a positive regulator of H5N1 replication and might serve as an alternative target for anti-influenza drug development.

Neuraminidase Inhibitor of Garcinia atroviridis L. Fruits and Leaves Using Partial Purification and Molecular Characterization.

Neuraminidase (NA) is an enzyme that prevents virions from aggregating within the host cell and promotes cell-to-cell spread by cleaving glycosidic linkages to sialic acid. The best-known neuraminidase is the viral neuraminidase, which present in the influenza virus. Thus, the development of anti-influenza drugs that inhibit NA has emerged as an important and intriguing approach in the treatment of influenza. Garcinia atroviridis L. (GA) dried fruits (GAF) are used commercially as seasoning and in beverages. The main objective of this study was to identify a new potential neuraminidase inhibitor from GA. A bioassay-guided fractionation method was applied to obtain the bioactive compounds leading to the identification of garcinia acid and naringenin. In an enzyme inhibition study, garcinia acid demonstrated the highest activity when compared to naringenin. Garcinia acid had the highest activity, with an IC50 of 17.34–17.53 µg/mL or 91.22–92.21 µM against Clostridium perfringens-NA, and 56.71–57.85 µg/mL or 298.32–304.31 µM against H1N1-NA. Based on molecular docking results, garcinia acid interacted with the triad arginine residues (Arg118, Arg292, and Arg371) of the viral neuraminidase, implying that this compound has the potential to act as a NA enzyme inhibitor.

Design, synthesis and biological evaluation of 1,3,4-triazole-3-acetamide derivatives as potent neuraminidase inhibitors

Neuraminidase (NA) is an ideal target for the development of anti-influenza drugs. In this paper, ZINC06057848 was screened out as a hit compound by docking-based virtual screening and molecular dynamics (MD) simulation. The modification and optimization of hit ZINC06057848 resulted in the discovery of a series of novel 1,3,4-triazole-containing NA inhibitors (5a-5j). Compound 5c exerts the best inhibitory activity (IC50 = 0.11 µM) against NA, which is comparable to the positive control oseltamivir carboxylate (OSC) (IC50 = 0.10 µM). Molecular docking analysis indicates that the good efficacy of inhibitor 5c may be attributed to the furan and triazole rings extending into 430-cavity and the ethylbenzene part occupying the active site. The results of this work may help in the development of new NA inhibitors.

Creation of a model for studying the antiviral effect of small interfering RNAs in vitro

Influenza is a widespread respiratory infection, accompanied by damage to the lower respiratory tract of a person, and can lead to severe complications, up to a fatal outcome. Currently existing therapeutic agents and vaccines do not provide fully effective protection against influenza viruses. The development and creation of drugs based on the mechanism of RNA interference in the context of this problem is a promising direction. The aim of this study is to select and experimentally substantiate cellular target genes for miRNAs whose knockdown suppresses viral reproduction. A549 cells (human lung adenocarcinoma) were transfected with small interfering RNAs. After 4 hours, the transfected cells were infected with the influenza virus pri MOI = 0.1; 0.01 and 0.001. The virus-containing liquid was sampled within three days from the moment of transfection and the intensity of the dynamics of viral reproduction was assessed by the CPD titration method. The use of all small interfering RNAs at MOI = 0.1 resulted in a significantly significant decrease in the viral titer relative to non-specific control. Similar results were obtained at MOI = 0.01 and 0.001. The most effective siRNA ISSINUP98, since when using it, at MOI = 0.1, the viral titer values decreased by 1.7 lg TCD50/ml and by 3 lg TCD50/ml at MOI = 0.01 on the third day. The results showed that miRNAs directed to human cellular genes FLT4, Nup98 and Nup205, whose derivatives play an important role in the life cycle of the influenza virus, effectively reduce its reproduction in vitro. Thus, the studied genes and their products are potential targets for the development of anti-influenza drugs.

Creation of a model for studying the antiviral effect of small interfering RNAs in vitro

Influenza is a widespread respiratory infection, accompanied by damage to the lower respiratory tract of a person, and can lead to severe complications, up to a fatal outcome. Currently existing therapeutic agents and vaccines do not provide fully effective protection against influenza viruses. The development and creation of drugs based on the mechanism of RNA interference in the context of this problem is a promising direction. The aim of this study is to select and experimentally substantiate cellular target genes for miRNAs whose knockdown suppresses viral reproduction. A549 cells (human lung adenocarcinoma) were transfected with small interfering RNAs. After 4 hours, the transfected cells were infected with the influenza virus pri MOI = 0.1; 0.01 and 0.001. The virus-containing liquid was sampled within three days from the moment of transfection and the intensity of the dynamics of viral reproduction was assessed by the CPD titration method. The use of all small interfering RNAs at MOI = 0.1 resulted in a significantly significant decrease in the viral titer relative to non-specific control. Similar results were obtained at MOI = 0.01 and 0.001. The most effective siRNA ISSINUP98, since when using it, at MOI = 0.1, the viral titer values decreased by 1.7 lg TCD50/ml and by 3 lg TCD50/ml at MOI = 0.01 on the third day. The results showed that miRNAs directed to human cellular genes FLT4, Nup98 and Nup205, whose derivatives play an important role in the life cycle of the influenza virus, effectively reduce its reproduction in vitro. Thus, the studied genes and their products are potential targets for the development of anti-influenza drugs.

The Natural Oligoribonucleotides Functionalized by D-Mannitol Affected Interactions of Hemagglutinin with Glycan Receptor Indicating Anti-Influenza Activity.

Hemagglutinin (HA), the class I influenza A virus protein is responsible for the attachment of virus particles to the cell by binding to glycan receptors, subsequent virion internalization, and cell entry. Consequently, the importance of HA makes it a primary target for the development of anti-influenza drugs. The natural oligoribonucleotides (ORNs) as well as their derivatives functionalized with D-mannitol (ORNs-D-M) possess anti-influenza properties in vitro and in vivo due to interaction with HA receptor sites. This activity suppresses the viral infection in host cells. In the present work, the complexes of ORNs and ORNs-D-M with HA protein were studied by agglutination assay, fluorescence spectroscopy, as well as molecular docking simulations. Acquired experimental data exhibited a decrease in HA titer by 32 times after incubation with the ORNs-D-M for 0.5–24 h. Quenching fluorescence intensity of the HA suggests that titration by ORNs and ORNs-D-M probably leads to changes in the HA structure. Detailed structural data were obtained with the molecular docking simulations performed for ORNs and ORNs-D-M ligands containing three and six oligoribonucleotides. The results reveal that a majority of the ORNs and ORNs-D-M bind in a non-specific way to the receptor-binding domain of the HA protein. The ligand’s affinity to the hemagglutinin was estimated at the micromolar level. Presented experimental data confirmed that both natural ORNs and functionalized ORNs-D-M inhibit the interactions between HA and glycan receptors and demonstrate anti-influenza activity.

A study on catalytic and non-catalytic sites of H5N1 and H1N1 neuraminidase as the target for chalcone inhibitors

The H1N1 pandemic in 2009 and the H5N1 outbreak in 2005 have shocked the world as millions of people were infected and hundreds of thousands died due to the infections by the influenza virus. Oseltamivir, the most common drug to block the viral life cycle by inhibiting neuraminidase (NA) enzyme, has been less effective in some resistant cases due to the virus mutation. Presently, the binding of 10 chalcone derivatives towards H5N1 and H1N1 NAs in the non-catalytic and catalytic sites was studied using molecular docking. The in silico study was also conducted for its drug-like likeness such as Lipinski Rule, mutagenicity, toxicity and pharmacokinetic profiles. The result demonstrates that two chalcones (1c and 2b) have the potential for future NA inhibitor development. Compound 1c inhibits H5N1 NA and H1N1 NA with IC50 of 27.63 µM and 28.11 µM, respectively, whereas compound 2b inhibits NAs with IC50 of 87.54 µM and 73.17 µM for H5N1 and H1N1, respectively. The in silico drug-like likeness prediction reveals that 1c is 62% better than 2b (58%) in meeting the criteria. The results suggested that 1c and 2b have potencies to be developed as non-competitive inhibitors of neuraminidase for the future development of anti-influenza drugs.

The Role of Lipid Metabolism in Influenza A Virus Infection.

Influenza A virus (IAV) is an important zoonotic pathogen that can cause disease in animals such as poultry and pigs, and it can cause infection and even death in humans, posing a serious threat to public health. IAV is an enveloped virus that relies on host cell metabolic systems, especially lipid metabolism systems, to complete its life cycle in host cells. On the other side, host cells regulate their metabolic processes to prevent IAV replication and maintain their normal physiological functions. This review summarizes the roles of fatty acid, cholesterol, phospholipid and glycolipid metabolism in IAV infection, proposes future research challenges, and looks forward to the prospective application of lipid metabolism modification to limit IAV infection, which will provide new directions for the development of anti-influenza drugs.

KIF11 inhibition decreases cytopathogenesis and replication of influenza A virus

Seasonal flu is an infectious disease of the respiratory tract caused by influenza viruses. The development of anti-influenza drugs has significantly reduced the threat from the influenza virus; however, frequent mutations of this negative RNA virus result in antiviral-resistant strains, and constantly intimidate the human race. Thus, identifying novel therapeutic targets for the prevention and treatment of influenza virus infections is critical. We aimed to determine whether the kinesin superfamily protein 11 (KIF11) inhibitors, monastrol and K858, inhibit viral cytopathogenesis and influenza A virus (IAV) replication. When MDCK or HEK293 cells were treated with monastrol and K858 that did not induce significant cytotoxicity, IAV-induced cytopathic effect was attenuated significantly. Furthermore, these inhibitors effectively suppressed the production of viral RNA, proteins, and infectious viral particles. Inhibition of KIF11 activity effectively attenuates virus-mediated cytopathic effect and suppresses viral replication. Hence, KIF11 is a potential therapeutic target against the influenza virus.

Design, synthesis and biological evaluation of dihydrofurocoumarin derivatives as potent neuraminidase inhibitors

Neuraminidase (NA) is a promising target for development of anti-influenza drugs. In this study a dihydrofurocoumarin derivative ZINC05577497 was discovered as a lead NA inhibitor based on docking-based virtual screening technique. The optimization of lead ZINC05577497 led to the discovery of a series of novel NA inhibitors 5a-5j. Compound 5b has the most potent activity against NA with IC50 = 0.02 µM, which is lower than those of the reference oseltamivir carboxylate (OSC) (IC50 = 0.04 µM) and ZINC05577497 (IC50 = 0.11 µM). Other target compounds also show potential inhibition of NA activity. Molecular docking results indicate that the good potency of 5b may be attributed to the elongation of the dihydrofurocoumarin ring to the 150-cavity. The results of this paper will be useful to discover more potent NA inhibitors.

Development and Effects of Influenza Antiviral Drugs.

Influenza virus is a highly contagious zoonotic respiratory disease that causes seasonal outbreaks each year and unpredictable pandemics occasionally with high morbidity and mortality rates, posing a great threat to public health worldwide. Besides the limited effect of vaccines, the problem is exacerbated by the lack of drugs with strong antiviral activity against all flu strains. Currently, there are two classes of antiviral drugs available that are chemosynthetic and approved against influenza A virus for prophylactic and therapeutic treatment, but the appearance of drug-resistant virus strains is a serious issue that strikes at the core of influenza control. There is therefore an urgent need to develop new antiviral drugs. Many reports have shown that the development of novel bioactive plant extracts and microbial extracts has significant advantages in influenza treatment. This paper comprehensively reviews the development and effects of chemosynthetic drugs, plant extracts, and microbial extracts with influenza antiviral activity, hoping to provide some references for novel antiviral drug design and promising alternative candidates for further anti-influenza drug development.

Influenza A Viruses Enter Host Cells via Extracellular Ca2+ Influx-Involved Clathrin-Mediated Endocytosis.

Influenza A virus (IAV) is internalized into its host cells by endocytosis, which involves many cellular proteins and molecules. In this study, we focus on the function of calcium ion (Ca2+) in IAV endocytosis. We have found that IAV infection is accompanied by the increase in concentration of cytosolic Ca2+, which is mainly attributed to the influx of extracellular Ca2+. When Ca2+ influx is abolished, IAV internalization will be markedly suppressed, but the virus attachment to its host cells will be unaffected. Extracellular Ca2+ influx is essential to the clathrin-mediated endocytosis (CME) of IAVs but dispensable to the clathrin-independent endocytosis of the virus and is dispensable to the CME of transferrin or low-density lipoprotein as a control. Ca2+ influx might participate in the dynamin-promoted membrane fission in the CME of IAVs. Our study highlights that IAVs enter host cells via extracellular Ca2+ influx-involved clathrin- and dynamin-dependent endocytosis, which will facilitate better understanding of IAV infection and development of anti-influenza drugs.