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Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
Published on: October 8, 2010
Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy
Bishnupriya Bhattacharya1, Robin A Weiss, Clare Davis
1Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, Medical School, Royal Free and University College London, 46 Cleveland Street, London W1T 4JF, UK.
Insights
This study introduces a new method for directly visualizing human immunodeficiency virus type-1 (HIV-1) particles using confocal microscopy. This technique accurately identifies and quantifies HIV-1, offering a simpler alternative to electron microscopy.
Area of Science:
- Virology
- Microscopy
- Immunology
Background:
- Direct visualization and accurate quantification of human immunodeficiency virus type-1 (HIV-1) are crucial for understanding viral dynamics and developing effective treatments.
- Traditional methods like electron microscopy (EM) can be complex and time-consuming.
- There is a need for simpler, yet reliable, techniques for HIV-1 identification and quantitation.
Purpose of the Study:
- To develop and validate a novel method for the direct visualization and quantification of HIV-1 particles.
- To assess the utility of laser scanning confocal microscopy as an alternative to electron microscopy for HIV-1 detection.
- To correlate confocal microscopy counts with established methods like EM and quantitative PCR for HIV-1 RNA load.
Main Methods:
- Human immunodeficiency virus type-1 (HIV-1) containing samples were adsorbed onto a plastic surface.
- Samples were doubly labeled with antibodies specific for viral proteins and nucleic acid dyes.
- Laser scanning confocal microscopy was employed to detect the co-localization of viral proteins and nucleic acids.
Main Results:
- The technique allowed for specific identification of HIV-1 by detecting co-localization of viral proteins and nucleic acids.
- Eight different HIV-1 sub-types and three HIV-1 groups were quantified in infected peripheral blood mononuclear cell (PBMC) supernatants.
- Confocal microscopy counts showed strong correlation with electron microscopy (EM) counts and HIV-1 RNA loads determined by quantitative PCR.
Conclusions:
- Laser scanning confocal microscopy provides a direct and specific method for visualizing HIV-1 particles.
- This technique offers a simple and effective alternative to electron microscopy for HIV-1 identification and quantitation.
- The method's accuracy is supported by its strong correlation with established quantitative measures of HIV-1.
Abstract:
A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation.
