Fluorescently labeled virus

Noble, R. T., and Fuhrman, J. A. (2000). Rapid virus production and removal as measured with fluorescently labeled viruses as tracers. Appl. Environ. Microbiol. 66, 3790—3797. 

The detection of HIV-related proteins is one of the most challenging tasks. This is especially true because AIDS should be diagnosed as early as possible to enable an early and effective therapy of this infection. Pavski and Le (57) used the aptamer strategy to detect reverse transcriptase (RT) of the type 1 human immunodeficiency virus (HIV-1). A direct and specific ACE method was proposed using laser-induced fluorescence (ACE/LIF) as detection principle. Single-stranded DNA aptamers as probes fluorescently labeled were synthesized. The resulting aptamer is specific for HIV-1 RT, and it exhibited no cross-reactivity with RTs of the enhanced avian myeloblastosis virus (AMV), the Moloney murine leukemia virus (MMLV), or denatured HIV-1 RT. An affinity complex of RT 26-HIV-l RT was stable, with calibration curves linear up to 50 nM (6 /xg/mL) HIV-1 RT concentration. Both... 

Hybridisation can thus be used to produce probes, for example, for the detection of oligonucleotides in chick embryo tissue or of Epstein-Barr viruses (glandular fever) in transplant recipients - by detection of specific RNA strands. DNA dendrimers and corresponding detection devices have meanwhile become commercially available (Genisphere company). Fluorescence-labelled polynucleotide dendrimers have also been used for signal intensification in DNA microchip technology [59]. 

Figure 9.8 Bilayers and a mixed monolayer of biotinylated and fluorescently labeled Cowpea mosaic virus (CPMV) particles on gold slides imaged via fluorescence microscopy The bilayers are shown on the left and the mixed monolayer on the right. A schematic representation of the CPMV layer...

Certain viruses and bacterial toxins are endocytosed by a specialized pathway that involves invaginations of the plasma membrane known as caveolae. Recent studies have traced out this caveolar endocytic pathway by jointly tagging the cellular machinery with GFP and labeling viruses or bacterial toxins with fluorescent dyes, which enables their dual visualization in real time (23, 24). Such work has revealed several unanticipated properties of this pathway, such as the ability of viruses to induce formation of actin comets that propel virus-containing vesicles (23) and the stable, immobile nature of the caveolar coat that encases these vesicles (24). 

The use of monosaccharide-based arrays to detect bacterial toxins, viruses and bacteria has been reported [219]. Arrays of IV-acetyl galactosamine and IV-acetylneuraminic acid derivatives were immobilized and were probed with fluorescently labeled bacterial cells and protein... 

While no real labels meet all of these needs, the properties of some of the more recently introduced labelling systems are approaching the ideal. Radioisotopes, once the only type of label used for immunoassays, have clearly been overwhelmed by current applications of fluorescent labeling methods, enzyme labels, and even coenzyme and prosthetic group labels. A variety of alternative labels has also been investigated, including red blood cells, latex particles, viruses, metals, and free radicals. Table 6.1 shows a representative listing of labels used in modem immunoassays.1... 

Fluorescent Staining. Certain methods of tissue staining differentially detect the presence of particular chemical components in cells. One approach is to use a fluorescent label to trace components inside the cell. Fluorescent staining not only attests to the presence or absence of certain components in cells but also provides their precise localization in a particular cell or tissue type. For example, immunochem-istry uses fluorescent antibodies that specifically bind with the target structures in the tissue so that the labeled antibodies will highlight the presence of the structures of interest in the analyzed specimen. Some applications of these techniques are staining of virus-infected cells in tissues and detection of immune-complex deposition in the kidneys and skin of patients with lupus. 

Figure 2.20 Images of fluorescently labeled biopolymers in an isotropic solvent (left) and a nematic solvent (right) offd virus [94], Images (a)-(d) are, respectively, of actin, wormlike...

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

---