In Vitro Evaluation of Synergistic Inhibitory Effects of Neuraminidase Inhibitors and Methylglyoxal Against Influenza Virus Infection

ABSTRACTFerrets represent an invaluable animal model to study influenza virus pathogenesis and transmission. To further characterize this model, we developed a differentiated primary ferret nasal epithelial cell (FNEC) culture model for investigation of influenza A virus infection and virus-host interactions. This well-differentiated culture consists of various cell types, a mucociliary clearance system, and tight junctions, representing the nasal ciliated pseudostratified respiratory epithelium. Both α2,6-linked and α2,3-linked sialic acid (SA) receptors, which preferentially bind the hemagglutinin (HA) of human and avian influenza viruses, respectively, were detected on the apical surface of the culture with different cellular tropisms. In accordance with the distribution of SA receptors, we observed that a pre-2009 seasonal A(H1N1) virus infected both ciliated and nonciliated cells, whereas a highly pathogenic avian influenza (HPAI) A(H5N1) virus primarily infected nonciliated cells. Transmission electron microscopy revealed that virions were released from or associated with the apical membranes of ciliated, nonciliated, and mucin-secretory goblet cells. Upon infection, the HPAI A(H5N1) virus replicated to titers higher than those of the human A(H1N1) virus at 37°C; however, replication of the A(H5N1) virus was significantly attenuated at 33°C. Furthermore, we found that infection with the A(H5N1) virus induced higher expression levels of immune mediator genes and resulted in more cell damage/loss than with the human A(H1N1) virus. This primary differentiated FNEC culture model, recapitulating the structure of the nasal epithelium, provides a useful model to bridgein vivoandin vitrostudies of cellular tropism, infectivity, and pathogenesis of influenza viruses during the initial stages of infection.IMPORTANCEAlthough ferrets serve as an important model of influenza virus infection, much remains unknown about virus-host interactions in this species at the cellular level. The development of differentiated primary cultures of ferret nasal epithelial cells is an important step toward understanding cellular tropism and the mechanisms of influenza virus infection and replication in the airway milieu of this model. Using lectin staining and microscopy techniques, we characterized the sialic acid receptor distribution and the cellular composition of the culture model. We then evaluated the replication of and immune response to human and avian influenza viruses at relevant physiological temperatures. Our findings offer significant insight into this first line of defense against influenza virus infection and provide a model for the evaluation of emerging influenza viruses in a well-controlledin vitroenvironmental setting.

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Dendritic Cells Targeting Lactobacillus plantarum Strain NC8 with a Surface-Displayed Single-Chain Variable Fragment of CD11c Induce an Antigen-Specific Protective Cellular Immune Response

Infection and Immunity ◽

10.1128/iai.00759-19 ◽

2019 ◽

Vol 88 (2) ◽

Author(s):

Jing Liu ◽

Guilian Yang ◽

Haibin Huang ◽

Chunwei Shi ◽

Xing Gao ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Influenza Virus ◽

Virus Infection ◽

Lactobacillus Plantarum ◽

Influenza Virus Infection ◽

Cell Culture Supernatant ◽

Single Chain ◽

Ifn Γ

ABSTRACT Influenza A virus (H1N1) is an acute, highly contagious respiratory virus. The use of lactic acid bacteria (LAB) to deliver mucosal vaccines against influenza virus infection is a research hot spot. In this study, two recombinant Lactobacillus plantarum strains expressing hemagglutinin (HA) alone or coexpressing aCD11c-HA to target HA protein to dendritic cells (DCs) by fusion to an anti-CD11c single-chain antibody (aCD11c) were constructed. The activation of bone marrow dendritic cells (BMDCs) by recombinant strains and the interaction of activated BMDCs and sorted CD4+ or CD8+ T cells were evaluated through flow cytometry in vitro, and cellular supernatants were assessed by using an enzyme-linked immunosorbent assay kit. The results demonstrated that, compared to the HA strain, the aCD11c-HA strain significantly increased the activation of BMDCs and increased the production of CD4+ gamma interferon-positive (IFN-γ+) T cells, CD8+ IFN-γ+ T cells, and IFN-γ in the cell culture supernatant in vitro. Consistent with these results, the aCD11c-HA strain clearly increased the activation and maturation of DCs, the HA-specific responses of CD4+ IFN-γ+ T cells, CD8+ IFN-γ+ T cells, and CD8+ CD107a+ T cells, and the proliferation of T cells in the spleen, finally increasing the levels of specific antibodies and neutralizing antibodies in mice. In addition, the protection of immunized mice was observed after viral infection, as evidenced by improved weight loss, survival, and lung pathology. The adoptive transfer of CD8+ T cells from the aCD11c-HA mice to NOD/Lt-SCID mice resulted in a certain level of protection after influenza virus infection, highlighting the efficacy of the aCD11c targeting strategy.

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In vitro inhibition of influenza virus infection by a crude extract from Isatis indigotica root resulting in the prevention of viral attachment

Molecular Medicine Reports ◽

10.3892/mmr.2011.709 ◽

2011 ◽

Cited By ~ 3

Author(s):

Nanshan Zhong

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Crude Extract ◽

Influenza Virus Infection ◽

Isatis Indigotica ◽

Viral Attachment ◽

In Vitro Inhibition

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Nanobodies With In Vitro Neutralizing Activity Protect Mice Against H5N1 Influenza Virus Infection

The Journal of Infectious Diseases ◽

10.1093/infdis/jiq168 ◽

2011 ◽

Vol 203 (8) ◽

pp. 1063-1072 ◽

Cited By ~ 48

Author(s):

Lorena Itatí Ibañez ◽

Marina De Filette ◽

Anna Hultberg ◽

Theo Verrips ◽

Nigel Temperton ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza Virus Infection ◽

H5n1 Influenza Virus ◽

Neutralizing Activity ◽

H5n1 Influenza

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Mouse Saliva Inhibits Transit of Influenza Virus to the Lower Respiratory Tract by Efficiently Blocking Influenza Virus Neuraminidase Activity

Journal of Virology ◽

10.1128/jvi.00145-17 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 6

Author(s):

Brad Gilbertson ◽

Wy Ching Ng ◽

Simon Crawford ◽

Jenny L. McKimm-Breschkin ◽

Lorena E. Brown

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Respiratory Tract ◽

Active Site ◽

Lower Respiratory Tract ◽

Mdck Cells ◽

Influenza Virus Infection ◽

Natural Form ◽

Influenza Virus Neuraminidase

ABSTRACT We previously identified a novel inhibitor of influenza virus in mouse saliva that halts the progression of susceptible viruses from the upper to the lower respiratory tract of mice in vivo and neutralizes viral infectivity in MDCK cells. Here, we investigated the viral target of the salivary inhibitor by using reverse genetics to create hybrid viruses with some surface proteins derived from an inhibitor-sensitive strain and others from an inhibitor-resistant strain. These viruses demonstrated that the origin of the viral neuraminidase (NA), but not the hemagglutinin or matrix protein, was the determinant of susceptibility to the inhibitor. Comparison of the NA sequences of a panel of H3N2 viruses with differing sensitivities to the salivary inhibitor revealed that surface residues 368 to 370 (N2 numbering) outside the active site played a key role in resistance. Resistant viruses contained an EDS motif at this location, and mutation to either EES or KDS, found in highly susceptible strains, significantly increased in vitro susceptibility to the inhibitor and reduced the ability of the virus to progress to the lungs when the viral inoculum was initially confined to the upper respiratory tract. In the presence of saliva, viral strains with a susceptible NA could not be efficiently released from the surfaces of infected MDCK cells and had reduced enzymatic activity based on their ability to cleave substrate in vitro. This work indicates that the mouse has evolved an innate inhibitor similar in function, though not in mechanism, to what humans have created synthetically as an antiviral drug for influenza virus. IMPORTANCE Despite widespread use of experimental pulmonary infection of the laboratory mouse to study influenza virus infection and pathogenesis, to our knowledge, mice do not naturally succumb to influenza. Here, we show that mice produce their own natural form of neuraminidase inhibitor in saliva that stops the virus from reaching the lungs, providing a possible mechanism through which the species may not experience severe influenza virus infection in the wild. We show that the murine salivary inhibitor targets the outer surface of the influenza virus neuraminidase, possibly occluding entry to the enzymatic site rather than binding within the active site like commercially available neuraminidase inhibitors. This knowledge sheds light on how the natural inhibitors of particular species combat infection.

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Inhibitory effects of carbocisteine on type A seasonal influenza virus infection in human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00376.2009 ◽

2010 ◽

Vol 299 (2) ◽

pp. L160-L168 ◽

Cited By ~ 11

Author(s):

Mutsuo Yamaya ◽

Hidekazu Nishimura ◽

Kyoko Shinya ◽

Yukimasa Hatachi ◽

Takahiko Sasaki ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Virus Infection ◽

Seasonal Influenza ◽

Influenza Virus Infection ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Human Influenza ◽

Inhibitory Effects ◽

Human Influenza Virus

Type A human seasonal influenza (FluA) virus infection causes exacerbations of bronchial asthma and chronic obstructive pulmonary disease (COPD). l-carbocisteine, a mucolytic agent, reduces the frequency of common colds and exacerbations in COPD. However, the inhibitory effects of l-carbocisteine on FluA virus infection are uncertain. We studied the effects of l-carbocisteine on FluA virus infection in airway epithelial cells. Human tracheal epithelial cells were pretreated with l-carbocisteine and infected with FluA virus (H3N2). Viral titers in supernatant fluids, RNA of FluA virus in the cells, and concentrations of proinflammatory cytokines in supernatant fluids, including IL-6, increased with time after infection. l-carbocisteine reduced viral titers in supernatant fluids, RNA of FluA virus in the cells, the susceptibility to FluA virus infection, and concentrations of cytokines induced by virus infection. The epithelial cells expressed sialic acid with an α2,6-linkage (SAα2,6Gal), a receptor for human influenza virus on the cells, and l-carbocisteine reduced the expression of SAα2,6Gal. l-carbocisteine reduced the number of acidic endosomes from which FluA viral RNA enters into the cytoplasm and reduced the fluorescence intensity from acidic endosomes. Furthermore, l-carbocisteine reduced NF-κB proteins including p50 and p65 in the nuclear extracts of the cells. These findings suggest that l-carbocisteine may inhibit FluA virus infection, partly through the reduced expression of the receptor for human influenza virus in the human airway epithelial cells via the inhibition of NF-κB and through increasing pH in endosomes. l-carbocisteine may reduce airway inflammation in influenza virus infection.

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