Isolation and quantitation of HIV in peripheral blood
Richard A Koup1, David D Ho1, Guido Poli2
1Aaron Diamond AIDS Research Center and New York University School of Medicine, New York, New York.
Insights
Quantifying replication-competent human immunodeficiency virus (HIV) in blood is vital for understanding HIV pathogenesis and therapy. This study details methods for determining HIV titer using tissue culture infectious dose (TCID) and evaluating CD4+ cell depletion.
Area of Science:
- Virology
- Immunology
- Infectious Diseases
Background:
- Quantifying replication-competent human immunodeficiency virus (HIV) in peripheral blood is essential for HIV pathogenesis and therapy research.
- HIV infection leads to CD4+ T cell depletion, a key indicator of disease progression.
Purpose of the Study:
- To establish reliable methods for quantifying infectious HIV in patient samples.
- To provide protocols for assessing HIV's impact on CD4+ T cells.
Main Methods:
- Determining HIV titer via end-point dilution to measure tissue culture infectious dose (TCID).
- Utilizing PHA-stimulated T cell blasts in co-culture for short-term HIV growth.
- Employing the Reed-Muench equation for TCID(50) calculation with multiple replicates.
- Evaluating cytopathic effects, including syncytia formation and single-cell death, to assess CD4+ cell depletion.
Main Results:
- The study outlines a basic protocol for HIV titer determination using TCID.
- An alternate protocol is presented for calculating TCID(50) using the Reed-Muench equation.
- Methods for evaluating HIV-induced cytopathic effects on CD4+ cells are detailed.
Conclusions:
- The provided protocols enable accurate quantitation of infectious HIV in peripheral blood.
- These methods are crucial for monitoring HIV progression and evaluating therapeutic efficacy.
- Assessing cytopathic effects offers insights into the mechanisms of CD4+ T cell depletion during HIV infection.
Abstract:
Quantitation of replication-competent human immunodeficiency virus (HIV) in peripheral blood of infected individuals is critical for investigations of HIV pathogenesis and therapy. In this unit, the basic protocol determines the HIV titer in seropositive blood by measuring the tissue culture infectious dose (TCID) by an end-point dilution method. A second basic protocol utilizes the PHA-stimulated T cell blasts (activated T cells) in co-culture with PBMC as described in the first basic protocol for the short-term growth of HIV in vitro. An Alternate Protocol describes the accumulative method of determining 50% tissue culture infectious dose (TCID(50)) of HIV using the Reed-Muench equation when multiple replicates of a given sample are employed in the assay. A consequence of HIV infection is the depletion of CD4(+) target cells, evidenced by syncytia formation or single-cell death; two support protocols detail the evaluation of these cytopathic effects.
