title: HIV-1 isolation from infected peripheral blood mononuclear cells.
abstract: Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.
authors.affiliation: Viral Evolution and Transmission Unit, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Dispinseri
authors.forename: Elisa
authors.initials: E
authors.lastname: Saba
authors.forename: Elisa
authors.initials: E
authors.lastname: Vicenzi
authors.forename: Neeltje A
authors.initials: NA
authors.lastname: Kootstra
authors.forename: Hanneke
authors.initials: H
authors.lastname: Schuitemaker
authors.forename: Gabriella
authors.initials: G
authors.lastname: Scarlatti
mesh_terms: D058621:Blood Buffy Coat / Q000821:virology; D016655:HIV Core Protein p24 / Q000378:metabolism; D015658:HIV Infections / Q000097:blood*; D015497:HIV-1 / Q000201:enzymology / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D016655:HIV Core Protein p24; D012194:RNA-Directed DNA Polymerase