Vigna radiata (L.) R. Wilczek Extract Inhibits Influenza A Virus by Targeting Viral Attachment, Penetration, Assembly, and Release

Vigna radiata (L.) R. Wilczek (mung bean) is a Chinese functional food with antioxidant, antimicrobial and anti-inflammatory activities. However, little is known about its antiviral activity. We aimed to investigate the antiviral activity and mechanisms of action of Vigna radiata extract (VRE) against influenza virus. HPLC was conducted to analyze the components of the VRE. The anti-influenza viral activity of VRE in Mardin-Darby canine kidney (MDCK) cells was evaluated by virus titration assays, hemagglutination assays, quantitative RT-PCR assays, cellular α-glucosidase activity assays and neuraminidase activity assays. Chromatographic profiling analysis identified two major flavonoids, vitexin and isovitexin, in the ethanol extract of Vigna radiata. Through in vitro studies, we showed that VRE, at concentrations up to 2,000 μg/ml, exhibited no cytotoxicity in MDCK cells. VRE protected cells from influenza virus-induced cytopathic effects and significantly inhibited viral replication in a concentration-dependent manner. A detailed time-of-addition assay revealed that VRE may act on both the early and late stages of the viral life cycle. We demonstrated that 1) VRE inhibits virus entry by directly blocking the HA protein of influenza virus; 2) VRE inhibits virus entry by directly binding to cellular receptors; 3) VRE inhibits virus penetration; 4) VRE inhibits virus assembly by blocking cellular α-glucosidase activity, thus reducing HA protein trafficking to the cell surface; and 5) VRE inhibits virus release by inhibiting viral neuraminidase activity. In summary, Vigna radiata extract potently interferes with two different subtypes of influenza viruses at multiple steps during the infectious cycle, demonstrating its broad-spectrum potential as an anti-influenza preventive and therapeutic agent. Continued development of Vigna radiata-derived products into antiviral therapeutics is warranted.

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Antiviral activity of novel oseltamivir derivatives against some influenza virus strains.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1872 ◽

2014 ◽

Vol 61 (3) ◽

Cited By ~ 2

Author(s):

Janusz Kocik ◽

Marcin Kołodziej ◽

Justyna Joniec ◽

Magdalena Kwiatek ◽

Michał Bartoszcze

Keyword(s):

Influenza Virus ◽

Antiviral Activity ◽

Mdck Cells ◽

Antiviral Effect ◽

In Vitro Cytotoxicity ◽

Influenza Viruses ◽

H3n2 Virus ◽

High Concentration ◽

Virus Dose

The aim of this study was to investigate the in vitro cytotoxicity of oseltamivir derivatives and determine their activity against A/H1N1/PR/8/34 and A/H3N2/HongKong/8/68 - strains of influenza virus. Antiviral activity of these compounds was determined by using two methods. MTT staining was used to assess the viability of MDCK cells infected with influenza viruses and treated with various concentrations of drugs. In parallel, the effect of drugs on viral replication was assessed using the hemagglutination test. The most toxic compounds were: OS-64, OS-35, OS-29, OS-27 and OS-25, whereas OS-11, OS-20 and OS-23 were the least toxic ones. Statistically significant antiviral effect at a higher virus dose was shown by compounds: OS-11, OS-20, OS-27, OS-35, and OS-64. H3N2 virus was sensitive to 10-times lower concentrations of OS-11 and OS-35 than H1N1. At a lower infection dose, the antiviral activity was observed for OS-11, OS 27, OS-35 and OS-20. OS-64 turned out to be effective only at a high concentration. OS-23 showed no antiviral effect.

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A Study on Host Tropism Determinants of Influenza Virus Using Machine Learning

Current Bioinformatics ◽

10.2174/1574893614666191104160927 ◽

2020 ◽

Vol 15 (2) ◽

pp. 121-134 ◽

Cited By ~ 2

Author(s):

Eunmi Kwon ◽

Myeongji Cho ◽

Hayeon Kim ◽

Hyeon S. Son

Keyword(s):

Machine Learning ◽

Amino Acids ◽

Influenza Virus ◽

Random Forest ◽

Physicochemical Properties ◽

Protein Sequences ◽

Influenza Viruses ◽

Host Tropism ◽

Post Hoc ◽

Ha Protein

Background: The host tropism determinants of influenza virus, which cause changes in the host range and increase the likelihood of interaction with specific hosts, are critical for understanding the infection and propagation of the virus in diverse host species. Methods: Six types of protein sequences of influenza viral strains isolated from three classes of hosts (avian, human, and swine) were obtained. Random forest, naïve Bayes classification, and knearest neighbor algorithms were used for host classification. The Java language was used for sequence analysis programming and identifying host-specific position markers. Results: A machine learning technique was explored to derive the physicochemical properties of amino acids used in host classification and prediction. HA protein was found to play the most important role in determining host tropism of the influenza virus, and the random forest method yielded the highest accuracy in host prediction. Conserved amino acids that exhibited host-specific differences were also selected and verified, and they were found to be useful position markers for host classification. Finally, ANOVA analysis and post-hoc testing revealed that the physicochemical properties of amino acids, comprising protein sequences combined with position markers, differed significantly among hosts. Conclusion: The host tropism determinants and position markers described in this study can be used in related research to classify, identify, and predict the hosts of influenza viruses that are currently susceptible or likely to be infected in the future.

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Synthesis of Four Pentacyclic Triterpene–Sialylglycopeptide Conjugates and Their Affinity Assays with Hemagglutinin

Molecules ◽

10.3390/molecules26040895 ◽

2021 ◽

Vol 26 (4) ◽

pp. 895

Author(s):

Mei Luo ◽

Ximin Wu ◽

Yiming Li ◽

Fujiang Guo

Keyword(s):

Influenza Virus ◽

Virus Entry ◽

Cycloaddition Reaction ◽

Pentacyclic Triterpene ◽

Entry Inhibitors ◽

Important Target ◽

Virus Entry Inhibitors ◽

Influenza Outbreaks ◽

Ha Protein ◽

Dissociation Constant Kd

Influenza outbreaks pose a serious threat to human health. Hemagglutinin (HA) is an important target for influenza virus entry inhibitors. In this study, we synthesized four pentacyclic triterpene conjugates with a sialylglycopeptide scaffold through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and prepared affinity assays of these conjugates with two HAs, namely H1N1 (A/WSN/1933) and H5N1 (A/Hong Kong/483/97), respectively. With a dissociation constant (KD) of 6.89 μM, SCT-Asn-betulinic acid exhibited the strongest affinity with the H1N1 protein. Furthermore, with a KD value of 9.10 μM, SCT-Asn-oleanolic acid exhibited the strongest affinity with the H5N1 protein. The conjugates considerably enhanced antiviral activity, which indicates that pentacyclic triterpenes can be used as a ligand to improve the anti-influenza ability of the sialylglycopeptide molecule by acting on the HA protein.

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Plasticity of the Influenza Virus H5 HA Protein

mBio ◽

10.1128/mbio.03324-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huihui Kong ◽

David F. Burke ◽

Tiago Jose da Silva Lopes ◽

Kosuke Takada ◽

Masaki Imai ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Influenza A ◽

Viral Antigen ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Antigenic Properties ◽

Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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Antiviral Activity of Benzoic Acid Derivative NC-5 Against Influenza A Virus and Its Neuraminidase Inhibition

International Journal of Molecular Sciences ◽

10.3390/ijms20246261 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6261

Author(s):

Min Guo ◽

Jiawei Ni ◽

Jie Yu ◽

Jing Jin ◽

Lingman Ma ◽

...

Keyword(s):

Antiviral Activity ◽

Benzoic Acid ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Influenza Viruses ◽

Drug Candidate ◽

Dependent Manner ◽

Drug Resistant

The currently available drugs against influenza A virus primarily target neuraminidase (NA) or the matrix protein 2 (M2) ion channel. The emergence of drug-resistant viruses requires the development of new antiviral chemicals. Our study applied a cell-based approach to evaluate the antiviral activity of a series of newly synthesized benzoic acid derivatives, and 4-(2,2-Bis(hydroxymethyl)-5-oxopyrrolidin-l-yl)-3-(5-cyclohexyl-4H-1,2,4-triazol-3-yl)amino). benzoic acid, termed NC-5, was found to possess antiviral activity. NC-5 inhibited influenza A viruses A/FM/1/47 (H1N1), A/Beijing/32/92 (H3N2) and oseltamivir-resistant mutant A/FM/1/47-H275Y (H1N1-H275Y) in a dose-dependent manner. The 50% effective concentrations (EC50) for H1N1 and H1N1-H275Y were 33.6 μM and 32.8 μM, respectively, which showed that NC-5 had a great advantage over oseltamivir in drug-resistant virus infections. The 50% cytotoxic concentration (CC50) of NC-5 was greater than 640 μM. Orally administered NC-5 protected mice infected with H1N1 and H1N1-H275Y, conferring 80% and 60% survival at 100 mg/kg/d, reducing body weight loss, and alleviating virus-induced lung injury. NC-5 could suppress NP and M1 protein expression levels during the late stages of viral biosynthesis and inhibit NA activity, which may influence virus release. Our study proved that NC-5 has potent anti-influenza activity in vivo and in vitro, meaning that it could be regarded as a promising drug candidate to treat infection with influenza viruses, including oseltamivir-resistant viruses.

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Characterization of a Porcine Lung Epithelial Cell Line Suitable for Influenza Virus Studies

Journal of Virology ◽

10.1128/jvi.75.19.9517-9525.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9517-9525 ◽

Cited By ~ 41

Author(s):

Sang Heui Seo ◽

Olga Goloubeva ◽

Richard Webby ◽

Robert G. Webster

Keyword(s):

Influenza Virus ◽

Epithelial Cell ◽

Cell Line ◽

Mdck Cells ◽

Influenza Viruses ◽

Lung Epithelial Cell ◽

Clinical Samples ◽

Epithelial Cell Line ◽

Porcine Endogenous Retrovirus ◽

Lung Epithelial

ABSTRACT We established a porcine lung epithelial cell line designated St. Jude porcine lung cells (SJPL) and demonstrated that all tested influenza A and B viruses replicated in this cell line. The infectivity titers of most viruses in SJPL cells were comparable to or better than those in MDCK cells. The propagation of influenza viruses from clinical samples in SJPL cells did not lead to antigenic changes in the hemagglutinin molecule. The numbers of both Sia2-3Gal and Sia2-6Gal receptors on SJPL cells were greater than those on MDCK cells. Influenza virus infection of SJPL cells did not lead to apoptosis, as did infection of MDCK cells. No porcine endogenous retrovirus was detected in SJPL cells, and in contrast to MDCK cells, SJPL cells did not cause tumors in nude mice.

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Discovery of Allopregnanolone Against Influenza Virus With Broad Spectrum in Vitro

10.21203/rs.3.rs-850522/v1 ◽

2021 ◽

Author(s):

yuqi Wang ◽

Yanyan Wang ◽

Hong Cao

Keyword(s):

Natural Products ◽

Influenza Virus ◽

Viral Replication ◽

Broad Spectrum ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Wild Type Virus ◽

Dependent Manner ◽

Viral Yield

Abstract Background: Influenza virus infection with seasonal or occasional but devastating morbidity and mortality, is a severe threat to public health. The frequent emergence of resistant viral strains limited application of current antivirals and posing an urgent need for novel antiviral therapies. Natural products offered a broad prospect in the screening and development of new influenza inhibitors.Methods: In this research, a high-throughput antiviral screening for 891 natural products was performed based on a recombinant reporter influenza A virus. According to the cytotoxicity assay and dose-response relationship, alloprogesterone (ALLO), as the positive hit was selected, and verified by viral titer reduction assay and immunofluorescence using a wild-type virus. Followingly, we explored its antiviral potency of counteracting with IAV and IBV, and preliminary investigated the mechanism of ALLO through time-of-addition assay and mini-replicon system.Results: Under the criteria of 80% inhibition and 70% cell viability, ALLO was screened out and confirmed antiviral activity in varied cells. The inhibitory effect of ALLO against influenza virus with a dose-dependent manner and significantly reduced viral yield of five different influenza viruses in the presence of 40 µM ALLO, including oseltamivir-resistant virus. Moreover, ALLO exhibited no influence on IAV entry or release during the viral replication cycle, but obviously interfered with the genome replication regarding post-infection 2 hrs to 6 hrs, which is consistent with the evidence of decreased polymerase activity.Conclusions: In summary, we firstly identified a new pharmacological activity of ALLO, as a broad spectrum inhibitor for treatment influenza infections, targeting viral replication stage and possessing great value of further development.

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Replication Dynamics, Pathogenicity, and Evolution of Influenza Viruses in Intestinal Caco-2 Cells

University of the Future: Re-Imagining Research and Higher Education ◽

10.29117/quarfe.2020.0166 ◽

2020 ◽

Author(s):

Israa Elbashir ◽

Heba Al Khatib ◽

Hadi Yassine

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Deep Sequencing ◽

Virus Evolution ◽

Influenza Viruses ◽

Intestinal Cells ◽

Sequencing Analysis ◽

Influenza Virus Replication ◽

Ha Protein ◽

Deep Sequencing Analysis

Background: Influenza virus is a major cause of respiratory infections worldwide. Besides the common respiratory symptoms, namouras cases with gastrointestinal symptoms have been reported. Moreover, influenza virus has been detected in feces of up to 20.6 % of influenza-infected patients. Therefore, direct infection of intestinal cells with influenza virus is suspected; however, the mechanism of this infection has not been explored. AIM: To investigate influenza virus replication, cellular responses to infection, and virus evolution following serial infection in human Caucasian colon adenocarcinoma cells (Caco-2 cells). Method: Two influenza A subtypes (A/H3N2 and A/H1N1pdm 09) and one influenza B virus (B/Yamagata) were serially passaged in Caco-2. Quantitative PCR was used to study hormones and cytokines expression following infection. Deep sequencing analysis of viral genome was used to assess the virus evolution. Results: The replication capacity of the three viruses was maintained throughout 12 passages, with H3N2 virus being the fastest in adaptation. The expression of hormone and cytokines in Caco-2 cells was considerably different between the viruses and among the passages, however, a pattern of induction was observed at the late phase of infection. Deep sequencing analysis revealed a few amino acid substitutions in the HA protein of H3N2 and H1N1 viruses, mostly in the antigenic site. Moreover, virus evolution at the quasispecies level based on HA protein revealed that H3N2 and H1N1 harbored more diverse virus populations when compared to IBV, indicating their higher evolution within Caco-2 cells. Conclusion: The findings of this study indicate the possibility of influenza virus replication in intestinal cells. To further explain the gastrointestinal complications of influenza infections in-vivo experiments with different influenza viruses are needed.

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Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus

Nutrients ◽

10.3390/nu13113911 ◽

2021 ◽

Vol 13 (11) ◽

pp. 3911

Author(s):

Ayaka Nakashima ◽

Yuka Horio ◽

Kengo Suzuki ◽

Yuji Isegawa

Keyword(s):

Influenza Virus ◽

Antiviral Activity ◽

Defense Mechanisms ◽

Mdck Cells ◽

Influenza Virus Infection ◽

Virus Titer ◽

Prolonged Treatment ◽

Action Mechanism ◽

Cell Defense ◽

Infected Cells

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin–Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

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Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01091-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Victoria Meliopoulos ◽

Sean Cherry ◽

Nicholas Wohlgemuth ◽

Rebekah Honce ◽

Karen Barnard ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Cell Lines ◽

Influenza A ◽

Vaccine Development ◽

Mdck Cells ◽

Influenza Viruses ◽

Clinical Samples ◽

Respiratory Epithelial Cells ◽

Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

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