An adenovirus-based fluorescent reporter vector to identify and isolate HIV-infected cells

2002 ◽  
Vol 99 (1-2) ◽  
pp. 9-21 ◽  
Author(s):  
Larry Richman ◽  
Pascal R.A Meylan ◽  
Miguel Munoz ◽  
Stéphane Pinaud ◽  
Jovan Mirkovitch
Keyword(s):  
Fluorescent Reporter ◽  
Reporter Vector ◽  
Infected Cells

scholarly journals More than efficacy revealed by single-cell analysis of antiviral therapeutics

2019 ◽  
Vol 5 (10) ◽  
pp. eaax4761 ◽  
Author(s):  
Wu Liu ◽  
Mehmet U. Caglar ◽  
Zhangming Mao ◽  
Andrew Woodman ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Viral Infection ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Principal Component ◽  
Cell Analysis ◽  
Infection Cycle ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Infection Dynamics ◽  
Infected Cells

Because many aspects of viral infection dynamics and inhibition are governed by stochastic processes, single-cell analysis should provide more information than approaches using population averaging. We have developed a microfluidic device composed of ~6000 wells, with each well containing a microstructure to capture single, infected cells replicating an enterovirus expressing a fluorescent reporter protein. We have used this system to characterize enterovirus inhibitors with distinct mechanisms of action. Single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates not only reveals efficacy but also facilitates clustering of drugs with the same mechanism of action and provides some indication of the ease with which resistance will develop.

scholarly journals Fluorescent Reporter Signals, EGFP, and DsRed, Encoded in HIV-1 Facilitate the Detection of Productively Infected Cells and Cell-Associated Viral Replication Levels

2012 ◽  
Vol 2 ◽  
Author(s):  
Kazutaka Terahara ◽  
Takuya Yamamoto ◽  
Yu-ya Mitsuki ◽  
Kentaro Shibusawa ◽  
Masayuki Ishige ◽  
...  
Keyword(s):  
Viral Replication ◽  
Fluorescent Reporter ◽  
Infected Cells ◽  
Hiv 1

scholarly journals QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 165 ◽  
Author(s):  
Lynda Handala ◽  
Tony Fiore ◽  
Yves Rouillé ◽  
Francois Helle
Keyword(s):  
Viral Infections ◽  
Java Virtual Machine ◽  
High Content Screening ◽  
Supplementary Data ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Processing Program ◽  
Infected Cells ◽  
Number Of Cells

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

scholarly journals Viral Mutagenesis as a Means for Generating Novel Proteins

2009 ◽  
Vol 84 (3) ◽  
pp. 1625-1630 ◽  
Author(s):  
John N. Davis ◽  
Anthony N. van den Pol
Keyword(s):  
Mammalian Cells ◽  
Protein A ◽  
Foreign Gene ◽  
Fluorescent Reporter ◽  
Novel Proteins ◽  
Gene Coding ◽  
New Variant ◽  
Infected Cells ◽  
Dsred Protein ◽  
Viral Mutagenesis

ABSTRACT We demonstrate that a mutation-prone virus engineered to express a foreign gene is an expedient means for generating novel mutant nonviral proteins in mammalian cells. Using vesicular stomatitis virus to express a gene coding for a fluorescent DsRed protein, a number of green mutant variants including a new variant not previously described were rapidly isolated from infected cells, sequenced, and cloned. Similar methods may be useful in the development of physiologically sensitive fluorescent reporter proteins and directed evolution or mutagenesis of proteins in general.

scholarly journals Infection with a newly-designed dual fluorescent reporter HIV-1 effectively identifies latently infected CD4+ T cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jinfeng Cai ◽  
Hongbo Gao ◽  
Jiacong Zhao ◽  
Shujing Hu ◽  
Xinyu Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Stable Cell Line ◽  
Fluorescent Reporter ◽  
Major Barrier ◽  
Latently Infected ◽  
Integration Sites ◽  
Compound Screening ◽  
Infected Cells ◽  
Hiv 1

The major barrier to curing HIV-1 infection is a small pool of latently infected cells that harbor replication-competent viruses, which are widely considered the origin of viral rebound when ART is interrupted. The difficulty of distinguishing latently infected cells from the vast majority of uninfected cells has represented a significant bottleneck precluding comprehensive understandings of HIV-1 latency. Here we reported and validated a newly-designed dual fluorescent reporter virus, DFV-B, infection with which in primary CD4+ T cells can directly label latently infected cells and generate a latency model that was highly physiological relevant. Applying DFV-B infection in Jurkat T cells, we generated a stable cell line model of HIV-1 latency with diverse viral integration sites. High-throughput compound screening with this model identified ACY-1215 as a potent latency reversing agent, which could be verified in other cell models and in primary CD4+ T cells from ART-suppressed individuals ex vivo. In summary, we have generated a meaningful and feasible model to directly study latently infected cells, which could open up new avenues to explore the critical events of HIV-1 latency and become a valuable tool for the research of AIDS functional cure.

Freeze-fracture, freeze-etch complementary pairs of virus-infected cells reveal value of etching even when relatively high concentrations of cryoprotectants are used

1978 ◽  
Vol 36 (2) ◽  
pp. 136-137
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Plant Tissue ◽  
Phosphate Buffer ◽  
Infected Plant ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Virus Infected Plant ◽  
Infected Cells ◽  
High Concentrations

It has been assumed by many involved in freeze-etch or freeze-fracture studies that it would be useless to etch specimens which were cryoprotected by more than 15% glycerol. We presumed that the amount of cryoprotective material exposed at the surface would serve as a contaminating layer and prevent the visualization of fine details. Recent unexpected freeze-etch results indicated that it would be useful to compare complementary replicas in which one-half of the frozen-fractured specimen would be shadowed and replicated immediately after fracturing whereas the complement would be etched at -98°C for 1 to 10 minutes before being shadowed and replicated.Standard complementary replica holders (Steere, 1973) with hinges removed were used for this study. Specimens consisting of unfixed virus-infected plant tissue infiltrated with 0.05 M phosphate buffer or distilled water were used without cryoprotectant. Some were permitted to settle through gradients to the desired concentrations of different cryoprotectants.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

Electron Microscopy of a Herpesvirus Isolated from Marek's Disease in Duck and Chicken Embryo Fibroblast Cultures

1968 ◽  
Vol 26 ◽  
pp. 222-223 ◽  
Author(s):  
Keyvan Nazerian
Keyword(s):  
Inclusion Bodies ◽  
Chicken Embryo Fibroblast ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Duck Embryo Fibroblast ◽  
Multinucleated Cells ◽  
Viral Particles ◽  
Infected Cells ◽  
And Control ◽  
Do So

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

Studies of a Defective Measles Virus

1968 ◽  
Vol 26 ◽  
pp. 208-209
Author(s):  
R. M. McCombs ◽  
M. Benyesh-Melnick ◽  
J. P. Brunschwig
Keyword(s):  
Inclusion Bodies ◽  
Measles Virus ◽  
Giant Cells ◽  
Helical Structures ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Culture Cells ◽  
Infected Cells ◽  
Eosinophilic Inclusions

Measles virus is an agent that is capable of replicating in a number of different culture cells and generally causes the formation of multinucleated giant cells. As a result of infection, virus is released from the cells into the culture fluids and reinfection can be initiated by this cell-free virus. The extracellular virus has been examined by negative staining with phosphotungstic acid and has been shown to be a rather pleomorphic particle with a diameter of about 140 mμ. However, no such virus particles have been detected in thin sections of the infected cells. Rather, the only virus-induced structures present in the giant cells are eosinophilic inclusions (intracytoplasmic or intranuclear). These inclusion bodies have been shown to contain helical structures, resembling the nucleocapsid observed in negatively stained preparations.

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