Development and application of monoclonal antibodies against avian influenza virus nucleoprotein

Yang, Ming; Berhane, Yohannes; Salo, Tim; Li, Mingyi; Hole, Kate; Clavijo, Alfonso

doi:10.1016/j.jviromet.2007.09.016

Cited by 60 publications

(50 citation statements)

References 16 publications

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“…After an additional 24 h (48 h after the initial transfection), the cells were infected with virus. The siRNA sequences are given in Table 1 and subjected to electrophoresis on 10% SDS-polyacrylamide gels (SDS-PAGE), transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore), and blotted with mouse monoclonal antibody to nonstructural protein 1 (anti-NS1) (3F5), mouse monoclonal antibody to nucleoprotein (anti-NP) (F26-9 [25]; gift from Mingyi Li), rabbit polyclonal antibody to hemagglutinin (anti-HA) (Rockland), mouse monoclonal anti-BAD (C-7; Santa Cruz), mouse monoclonal antibody against cleaved caspase-3 (anti-cleaved caspase-3) (Asp175; R&D Systems), rabbit monoclonal antibody against phosphorylated BAD (anti-phospho-BAD) (Ser136; Cell Signaling), mouse monoclonal anti-phospho-BAD (Ser112; Cell Signaling), rabbit polyclonal anti-cleaved PARP (Asp214; Cell Signaling), rabbit monoclonal anti-cytochrome c (Cell Signaling), rabbit polyclonal anti-cleaved caspase-7 (Asp198; Cell Signaling), or rabbit polyclonal anti-␤-actin (Cell Signaling) antibodies. Cytoplasmic lysate for caspase immunoblotting was obtained by lysing cells in caspase lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 100 M ␤-glycerol-3-phosphate, protease inhibitor cocktail [Roche]).…”

Section: Cellsmentioning

confidence: 99%

“…The membrane was probed for influenza virus proteins NS1, NP, and HA. The NS1 mouse monoclonal antibody was generated and characterized in our lab (M. N. Rahim, M. Selman, P. J. Sauder, W. Stecho, W. Xu, M. Lebar, E. G. Brown, and K. M. Coombs, submitted for publication), and the characterization of the mouse monoclonal NP antibody was previously described (25). In the stably expressing nontargeting shRNA control cells, NS1, NP, and HA viral proteins were detected as early as 4 hpi and strongly detected at 8 hpi onwards (Fig.…”

Section: Influenza Virus-induced Cytopathology and Cell Death Are Inhmentioning

confidence: 99%

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Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

Tran

Cortens

et al. 2013

J Virol

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e Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

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Section: Cellsmentioning

confidence: 99%

Section: Influenza Virus-induced Cytopathology and Cell Death Are Inhmentioning

confidence: 99%

Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

Tran

Cortens

et al. 2013

J Virol

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“…The PVDF membranes were briefly stained with Ponceau to confirm protein transfer, blocked with 5% skim milk, and probed with various antibodies. Primary antibodies were mouse anti-influenza NP protein (74), ␣-GAPDH, ␣-vimentin, ␣-␤-2-microglobulin, alpha vasodilatory-stimulated phosphoprotein (␣-VASP), rabbit anti-actin, ␣-Rock2, ␣-Akt, ␣-cytokeratin 10, ␣-Bid, and goat anti-poly(ADP-ribose) polymerase (PARP). Secondary antibodies were Alexa488-conjugated goat antimouse for NP and GAPDH, Alexa488-conjugated goat anti-rabbit for actin, or the appropriate horseradish peroxidase (HRP)-conjugated rabbit anti-mouse, goat anti-rabbit, or rabbit anti-goat for all other proteins.…”

Section: Cells and Viruses (I) Virusesmentioning

confidence: 99%

Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

Coombs

Berard

et al. 2010

J Virol

134

190

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Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular "transcriptome." Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways.

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“…Estes subtipos podem exterminar uma criação ou granja de aves em apenas 24 horas, pois se replicam sistematicamente nas aves infectadas, afetando não somente o trato respiratório como também o reprodutivo e gastrointestinal (YANG et al, 2008;SÁ;.…”

Section: Epidemiologia Patogenicidade E Transmissãounclassified

“…Além disso, através de estudos envolvendo o alinhamento de sequências de nucleotídeos e análises filogenéticas, Shu et al (1993) demonstraram que existe uma elevada similaridade entre as sequências de nucleotídeos dos genes das NPs de estipes do VIA, mesmo que isoladas de diferentes hospedeiros. Desta forma, por se tratar de uma proteína altamente conservada e que apresenta elevada imunogenicidade, a NP recombinante do VIA constitui-se um antígeno de eleição para ser aplicada no imunodiagnóstico da IA (CAPUA; ALEXANDER, 2002;MARTINS, 2001;HALVORSON, 2003;VARICH, 2014;YANG et al, 2008).…”

Section: Diagnóstico Sorológico Da Influenza Aviáriaunclassified