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.
Department of Microbiology, Keio University School of Medicine, Tokyo, Japan.
S
S
Kato
Y
Y
Hiraishi
N
N
Nishimura
T
T
Sugita
M
M
Tomihama
T
T
Takano
D002461:Cell Line, Transformed; D002478:Cells, Cultured; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016014:Linear Models; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010948:Viral Plaque Assay; D014771:Virion