A plaque hybridization assay for quantifying and cloning infectious human immunodeficiency virus type 1 virions

A biological method was developed for quantifying and cloning of infectious virions of human immunodeficiency virus type 1 (HIV-1). Virus preparations were mixed with permissive cells for binding, and the cells were cast in an agarose gel. After incubation for 9 days viral particles released from infected cells propagating from each initially infected cell were transferred on nylon membrane and subjected to hybridization using a radioactive HIV-1 DNA probe. Infectious centers of HIV-1 were detected as hybridization spots on autoradiographs regardless of cytopathic effects or syncytium formation. Three different CD4+ cell lines (MT-4, MOLT-4 and U937) and peripheral blood mononuclear cells from healthy donors were used as recipient cells. Infectious virions were recovered from a portion of agarose gel corresponding to each hybridization spot. This assay is suitable for quantifying infectious HIV-1 virions with different cell tropisms and for investigating the relationship between the phenotype and genotype of HIV-1 at a clonal level.