title: Superiority of infectivity-based over particle-based methods for quantitation of drug resistant HIV-1 as inocula for cell cultures.
abstract: Performance of phenotypic assays and replication capacity assays require normalization of virus input. Therefore, quantitation of HIV-1 in supernatants to inoculate cell cultures is an important step. Since the gold standard for the determination of infectivity, the tissue culture infectious dose 50% (TCID50) is time-consuming, several other methods are in use. This study evaluated methods for the quantitation of drug resistant viruses in cell culture supernatants. The compared methods were based on the detection of viral structural components like genomic RNA or p24 antigen (CA-p24) (particle-based), the determination of reverse transcriptase (RT) activity, and methods based on the detection of viral infectivity like LTR-induced beta-galactosidase (beta-gal) activity and the TCID50 (infectivity-based). Significant correlations were observed between beta-gal activity and TCID50, and between CA-p24 and viral RNA. RT activity did not correlate with any other method. However, RT activity correlated significantly with infectivity when non-resistant subtype-B isolates were analyzed. In contrast to viral infectivity, CA-p24 exhibited a long half life and accumulated in cell culture, resulting in decreasing ratios of infectious virions to CA-p24 over time. As a consequence, relative replication capacities of drug resistant viruses were only determined reliably if the input virus was normalized according to infectivity. In conclusion, RT activity seems to be feasible for non-resistant subtype-B viruses but may be of limited use for non-B subtypes and for drug resistant viruses. Methods determining infectivity are most suitable for quantitation of cell culture inocula, whereas particle-based assays are more appropriate for quantitation of virus production during an experiment.
authors.affiliation: Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, D-91054 Erlangen, Germany. monika.tschochner@viro.med.uni-erlangen.de
authors.forename: Monika
authors.initials: M
authors.lastname: Tschochner
authors.forename: Eva
authors.initials: E
authors.lastname: Schwingel
authors.forename: Christine
authors.initials: C
authors.lastname: Thein
authors.forename: Sabine
authors.initials: S
authors.lastname: Wittmann
authors.forename: Christiane
authors.initials: C
authors.lastname: Paatz
authors.forename: Hauke
authors.initials: H
authors.lastname: Walter
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D024882:Drug Resistance, Viral / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D017930:Genes, Reporter; D016679:Genome, Viral; D016655:HIV Core Protein p24 / Q000032:analysis*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000032:analysis*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D012367:RNA, Viral / Q000032:analysis; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D012367:RNA, Viral; D008156:Luciferases; D054303:HIV Reverse Transcriptase; D001616:beta-Galactosidase