title: A computer-based, image-analysis method to quantify HIV-1 infection in a single-cycle infectious center assay.
abstract: Rapid and precise quantitation of the infectivity of HIV is important for molecular virologic studies as well as for measuring the activities of antiviral drugs and neutralizing antibodies. In the present study, an indicator cell line and image-analysis software were used to quantify HIV infectivity. Cells of the P4R5 line, which express the receptors for HIV infection as well as beta-galactosidase under the control of the HIV-1 long terminal repeat, were infected either with CXCR4- or CCR5-using viruses, including primary isolates, then stained 2 days later with X-gal to turn infected cells blue. Digital images of monolayers of the infected cells were captured using a high resolution CCD video camera and a macro video zoom lens. A software program was developed to process the images and to count the blue-stained foci of infection. The assay was applied to assess the infectivity of site-directed viral mutants, and to measure the activity of antiviral drugs and neutralizing antibody. The results indicate that the described method allows for the rapid quantitation of infected cells over a wide range of viral inocula with reproducibility, accuracy and relatively low cost.
authors.affiliation: Department of Pathology, University of California San Diego, La Jolla, CA 92093-0679, USA. johnday88@yahoo.com
authors.forename: John R
authors.initials: JR
authors.lastname: Day
authors.forename: Laura E
authors.initials: LE
authors.lastname: Martínez
authors.forename: Roman
authors.initials: R
authors.lastname: Sásik
authors.forename: Douglas L
authors.initials: DL
authors.lastname: Hitchin
authors.forename: Megan E
authors.initials: ME
authors.lastname: Dueck
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Richman
authors.forename: John C
authors.initials: JC
authors.lastname: Guatelli
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D017930:Genes, Reporter; D015483:HIV Antibodies / Q000276:immunology; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D009154:Mutation; D009500:Neutralization Tests; D019829:Nevirapine / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000096:biosynthesis; D019718:Receptors, CXCR4 / Q000096:biosynthesis; D012984:Software; D001616:beta-Galactosidase / Q000032:analysis* / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D019829:Nevirapine; D001616:beta-Galactosidase