A microassay for determination of the cytopathogenicity of human immunodeficiency virus type-1 isolates.
Correlations between the in vitro biological properties of HIV strains isolated from patients and the prognosis of their disease have been reported. We developed a technique to study the phenotype of HIV strains isolated from patients. We used the P4 cell line, derived from HeLa cells, which has been transfected with receptor CD4 gene. HIV laboratory strain (HIVLAI) and peripheral blood leukocytes (PBLs) from donors infected with HIVLAI induce syncytium in P4 cell cultures in vitro. The presence of reporter gene (LacZ gene) under the control of the HIV-1 long terminal repeat (LTR) in these cells allows colorimetric visualization of syncytia in the cytoplasm using a beta-galactosidase (beta gal) assay in the presence of X-gal. We cocultivated 1 x 10(6) patient PBLs with 2 x 10(6) normal PHA-activated normal PBLs for 4 days in the presence of IL-2 in 24-well plates. Half of the medium was replaced twice a week and PHA-activated normal PBLs were added every 7 days. HIV-1 was isolated from cocultured PBLs of 18 patients with advanced-stage HIV infection as assessed by the production of HIV p24 detected with a commercially available HIV-1 p24 ELISA. Supernatant and 10(5) cells were collected twice a week from cocultured PBLs and were added to P4 cells in 96-well microtiter plates. The cultures were observed every day for 3 days and then the beta gal assay was performed. We did not observe any effect with cells and supernatant from 8 patients, harvested from cultures incubated for as long as 28 days. The phenotype of these isolates was called NC (noncytopathic). In cells from 2 patients, we obtained blue multinucleated giant cells; the phenotype of these strains was called SI (syncytium inducing). In cultures from 8 other patients, we obtained the death of P4 cells without syncytium formation, and the phenotype of these strains was called CI (cell-killing inducing). In every case, the cytopathic effect of HIV-1 isolates could be detected with cocultured PBLs collected as early as day 4 of culture. Cocultured PBLs from 13 healthy controls did not alter the P4 cells. We displayed the replication of CI strains of HIV-1, but not the one of NC strains in P4 cell line. Our micromethod allowed the detection of cytopathic effects of HIV isolates. Further investigations should define the clinical applications of this method.
Benyoucef
S
S
Laboratoire de Virologie, CHU, Lille, France.
Hober
D
D
Shen
L
L
Ajana
F
F
Gérard
Y
Y
Bocket-Mouton
L
L
Mouton
Y
Y
Wattré
P
P
D018920:Coculture Techniques; D003588:Cytopathogenic Effect, Viral; D005697:Galactosides; D015726:Giant Cells / Q000473:pathology* / Q000821:virology*; D015658:HIV Infections / Q000473:pathology* / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000302:isolation & purification* / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D009336:Necrosis; D014779:Virus Replication / Q000502:physiology
D005697:Galactosides