Isolation and Quantitation of HIV in Peripheral Blood

Richard A. Koup1, David D. Ho1, Guido Poli2, Anthony S. Fauci2

1 Aaron Diamond AIDS Research Center and New York University School of Medicine, New York, New York, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.2
DOI:  10.1002/0471142735.im1202s05
Online Posting Date:  May, 2001
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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 describes the accumulative method of determining 50% tissue culture infectious dose (TCID50) 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.

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Table of Contents

  • Basic Protocol 1: Culture of HIV in Peripheral Blood and Measurement of Titer by TCID Quantitation
  • Basic Protocol 2: Primary Isolation of HIV from PBMC by Infection of Allogeneic T Cell Blasts
  • Alternate Protocol 1: Assessment of HIV Titer Using the Reed‐Muench Accumulative Method
  • Support Protocol 1: Evaluation of the Cytopathic Effects of HIV on CD4+ Target Cells: Syncytia Formation
  • Support Protocol 2: Evaluation of the Cytopathic Effects of HIV on CD4+ Target Cells: HIV‐Mediated Single‐Cell Death
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Culture of HIV in Peripheral Blood and Measurement of Titer by TCID Quantitation

  • Whole blood from normal (HIV‐seronegative) and affected individuals (HIV‐seropositive)
  • Phosphate‐buffered saline (PBS), pH 7.2, or Hanks balanced salt solution (HBSS), sterile ( appendix 2A)
  • Ficoll‐Hypaque solution (unit 7.1)
  • Complete RPMI‐10 medium ( appendix 2A) supplemented with 2 µg/ul phytohemagglutinin (PHA) or 5% (v/v) human IL‐2 (Table 97.80.4711)
  • Evacuated tubes containing sodium heparin ( appendix 33)
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent), equipped with plate holders
  • 50‐ml polypropylene centrifuge tubes, sterile
  • 24‐well microtiter plates, sterile (Falcon #3047)
  • Additional reagents and equipment for blood collection ( appendix 33), Ficoll‐Hypaque gradients (unit 7.1), counting cells ( appendix 33), and determination of HIV activity by p24 antigen production (unit 12.5)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator.

Basic Protocol 2: Primary Isolation of HIV from PBMC by Infection of Allogeneic T Cell Blasts

  • T cell blasts isolated from HIV‐seronegative blood (first protocol 1basic protocol)
  • Peripheral blood mononuclear cells (PBMC) from HIV‐seropositive blood (first protocol 1basic protocol)
  • Complete RPMI‐10 medium ( appendix 2A), without and with 5% to 10% IL‐2 (depending on purity; Table 97.80.4711)
  • Tabletop centrifuge, refrigerated
  • Additional reagents and solutions for Ficoll‐Hypaque gradient centrifugation (unit 7.1), PHA‐induced proliferation of PBMC (unit 7.10), and determination of HIV activity by reverse transcriptase assays or p24 antigen production (unit 12.5)
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Literature Cited

Literature Cited
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Key Reference
   Ho, D.D., Yoshiyama, H., Mohri, H., Daar, E.S., and Cao, Y. 1991. Quantitation of HIV‐1: Significance in pathogenesis and therapy. In Viral Quantitation in HIV infection (J.M. Andrieu, ed.) pp. 3‐7. John Libbey Eurotext, Montrouge, France.
  Good overview of quantitative virology of HIV with examples of application to the study of pathogenesis and therapy.
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