Use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses.
A rapid and sensitive focal immunofluorescence assay (FIA) using monoclonal antibodies or heterologous antisera was employed for detection and biological cloning of viruses capable of inducing viral antigens on cell surfaces. The FIA was performed directly on a variety of live cells in tissue culture dishes and was used successfully with C-type murine leukemia viruses (MuLV) of different tropism including ecotropic, xenotropic, amphotropic, and dual-tropic recombinant mink cell focus-inducing (MCF) viruses. With the FIA, we were able to titrate and distinguish ecotropic Friend-MuLV and Friend-MCF viruses present in mixtures. Dual-tropic MCF viruses could be specifically detected directly in mouse cells by using MCF-specific monoclonal antibodies. These antibodies replaced the requirement for production of typical MCF cytopathic effect in mink cells for MCF virus detection, and also allowed efficient titration in mouse cells of MCF virions pseudotyped with ecotropic envelope proteins. Furthermore, by picking foci of fluorescent cells and using their cell-free viral progeny, MCF viruses were cloned from complex pseudotypic mixtures. This allowed the cloning of viruses present at low frequency in heterogeneous mixtures obtained from leukemic tissues.
Sitbon
M
M
Nishio
J
J
Wehrly
K
K
Lodmell
D
D
Chesebro
B
B
D000818:Animals; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D009052:Leukemia Virus, Murine / Q000254:growth & development*; D051379:Mice; D008907:Mink / Q000382:microbiology; D012190:Retroviridae / Q000032:analysis*; D013045:Species Specificity