Visualization of avian influenza virus infected cells using self-assembling fragments of green fluorescent protein

ABSTRACT The specificity of the CD4 T-cell immune response to influenza virus is influenced by the genetic complexity of the virus and periodic encounters with variant subtypes and strains. In order to understand what controls CD4 T-cell reactivity to influenza virus proteins and how the influenza virus-specific memory compartment is shaped over time, it is first necessary to understand the diversity of the primary CD4 T-cell response. In the study reported here, we have used an unbiased approach to evaluate the peptide specificity of CD4 T cells elicited after live influenza virus infection. We have focused on four viral proteins that have distinct intracellular distributions in infected cells, hemagglutinin (HA), neuraminidase (NA), nucleoprotein, and the NS1 protein, which is expressed in infected cells but excluded from virion particles. Our studies revealed an extensive diversity of influenza virus-specific CD4 T cells that includes T cells for each viral protein and for the unexpected immunogenicity of the NS1 protein. Due to the recent concern about pandemic avian influenza virus and because CD4 T cells specific for HA and NA may be particularly useful for promoting the production of neutralizing antibody to influenza virus, we have also evaluated the ability of HA- and NA-specific CD4 T cells elicited by a circulating H1N1 strain to cross-react with related sequences found in an avian H5N1 virus and find substantial cross-reactivity, suggesting that seasonal vaccines may help promote protection against avian influenza virus.

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Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01641-05 ◽

2006 ◽

Vol 50 (8) ◽

pp. 2806-2813 ◽

Cited By ~ 10

Author(s):

T. Ueno ◽

Y. Eizuru ◽

H. Katano ◽

T. Kurata ◽

T. Sata ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Line ◽

Real Time ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Fluorescent Protein ◽

Clinical Isolates ◽

Pml Bodies ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

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Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca2+ Influx, Leading to Apoptosis in Avian Cells

Journal of Virology ◽

10.1128/jvi.01923-09 ◽

2010 ◽

Vol 84 (6) ◽

pp. 3068-3078 ◽

Cited By ~ 37

Author(s):

Mayo Ueda ◽

Tomo Daidoji ◽

Anariwa Du ◽

Cheng-Song Yang ◽

Madiha S. Ibrahim ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Highly Pathogenic ◽

H5n1 Avian Influenza Virus ◽

H5n1 Avian Influenza ◽

Infected Cells ◽

Pathogenic H5n1

ABSTRACT In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca2+ dynamics in the infected cells and demonstrated an increase in the concentration of Ca2+ in the cytosol ([Ca2+]i) and mitochondria ([Ca2+]m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca2+ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca2+ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca2+, significantly reduced the [Ca2+]i, [Ca2+]m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca2+ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca2+ homeostasis on influenza A virus infection.

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Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Reproduction ◽

10.1530/rep-06-0233 ◽

2007 ◽

Vol 134 (3) ◽

pp. 445-453 ◽

Cited By ~ 23

Author(s):

Jiří Kalina ◽

Filip Šenigl ◽

Alena Mičáková ◽

Jitka Mucksová ◽

Jana Blažková ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Reporter Gene ◽

Fluorescent Protein ◽

Germ Line ◽

Retroviral Vector ◽

Male Germ Line ◽

Testicular Cells ◽

Infected Cells ◽

Green Fluorescent ◽

Transgenic Chickens

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated γ-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line.

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Hemagglutinin-Pseudotyped Green Fluorescent Protein-Expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.01433-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 2157-2163 ◽

Cited By ~ 73

Author(s):

Luis Martínez-Sobrido ◽

Richard Cadagan ◽

John Steel ◽

Christopher F. Basler ◽

Peter Palese ◽

...

Keyword(s):

Influenza Virus ◽

Green Fluorescent Protein ◽

Neutralizing Antibodies ◽

Fluorescent Protein ◽

Influenza Viruses ◽

Serum Samples ◽

Screening Assay ◽

Ha Gene ◽

Green Fluorescent ◽

Gene Expressing

ABSTRACT Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.

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Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

Journal of Virology ◽

10.1128/jvi.77.10.6070-6075.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 6070-6075 ◽

Cited By ~ 12

Author(s):

Dirk Spitzer ◽

Kurt E. J. Dittmar ◽

Manfred Rohde ◽

Hansjörg Hauser ◽

Dagmar Wirth

Keyword(s):

Green Fluorescent Protein ◽

Envelope Protein ◽

Fluorescent Protein ◽

Inverse Correlation ◽

Cell Interactions ◽

Virus Particles ◽

Viral Particles ◽

Infected Cells ◽

Env Protein ◽

Green Fluorescent

ABSTRACT Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.

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Human Cytomegalovirus Labeled with Green Fluorescent Protein for Live Analysis of Intracellular Particle Movements

Journal of Virology ◽

10.1128/jvi.79.5.2754-2767.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 2754-2767 ◽

Cited By ~ 96

Author(s):

Kerstin Laib Sampaio ◽

Yolaine Cavignac ◽

York-Dieter Stierhof ◽

Christian Sinzger

Keyword(s):

Green Fluorescent Protein ◽

Human Cytomegalovirus ◽

Fluorescent Protein ◽

Rare Event ◽

Single Step ◽

Wild Type Virus ◽

Cell Cortex ◽

Long Distance ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT Human cytomegalovirus (HCMV) replicates in the nuclei of infected cells. Successful replication therefore depends on particle movements between the cell cortex and nucleus during entry and egress. To visualize HCMV particles in living cells, we have generated a recombinant HCMV expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150). The resulting UL32-EGFP-HCMV was analyzed by immunofluorescence, electron microscopy, immunoblotting, confocal microscopy, and time-lapse microscopy to evaluate the growth properties of this virus and the dynamics of particle movements. UL32-EGFP-HCMV replicated similarly to wild-type virus in fibroblast cultures. Green fluorescent virus particles were released from infected cells. The fluorescence stayed associated with particles during viral entry, and fluorescent progeny particles appeared in the nucleus at 44 h after infection. Surprisingly, strict colocalization of pUL32 and the major capsid protein pUL86 within nuclear inclusions indicated that incorporation of pUL32 into nascent HCMV particles occurred simultaneously with or immediately after assembly of the capsid. A slow transport of nuclear particles towards the nuclear margin was demonstrated. Within the cytoplasm, most particles performed irregular short-distance movements, while a smaller fraction of particles performed centripetal and centrifugal long-distance movements. Although numerous particles accumulated in the cytoplasm, release of particles from infected cells was a rare event, consistent with a release rate of about 1 infectious unit per h per cell in HCMV-infected fibroblasts as calculated from single-step growth curves. UL32-EGFP-HCMV will be useful for further investigations into the entry, maturation, and release of this virus.

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Functional Analysis of the Human Cytomegalovirus US28 Gene by Insertion Mutagenesis with the Green Fluorescent Protein Gene

Journal of Virology ◽

10.1128/jvi.72.10.8158-8165.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8158-8165 ◽

Cited By ~ 88

Author(s):

Jeffrey Vieira ◽

Thomas J. Schall ◽

Lawrence Corey ◽

Adam P. Geballe

Keyword(s):

Green Fluorescent Protein ◽

Intracellular Calcium ◽

Human Cytomegalovirus ◽

Fluorescent Protein ◽

Early Gene ◽

Calcium Flux ◽

Northern Analysis ◽

Insertion Mutagenesis ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the β class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415–425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539–28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the β-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.

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