Infectivity Assays

As indicated above, the standard tumorigenesis assays were very cumbersome and time consuming limiting their usefulness. With EM showing VLP in both high and low cancer mouse strains, the mere presence or absence of particles was clearly not a surrogate breast tumorigenicity assay. Progress was accelerated somewhat as Nandi and De Ome introduced the "nodulo-genesis assay" that became a surrogate assay for infectivity (Nandi, 1963a,b). The assay used 3- to 5-week-old nulliparous female BALB/c mice that were injected intraperitoneally with the tissue, milk, or blood extract and stimulated with either subrenal capsule or subcutaneous transplants of pituitary glands to produce a pseudo-lactation. With removal of the pituitary transplants, the mammary glands regressed leaving the hyperplastic alveolar nodules (HAN). The HAN presence became the "marker" for MMTV infection. The process shortened detection assays from years to 4 months. Now, one could test for viral infectivity in a relatively short period.

Led by Etienne "Steve" Lassfargue, Moore's group explored tissue culture methods that had become popular with experiments using transforming agents such as Rous sarcoma virus (RSV), Polyomavirus (PyV), and other small DNA tumor viruses. However, the milk agent proved difficult to culture and even more difficult to detect. Even though Andervont had proven that transmission of the virus could be prevented by immunization using tissue extracts of tumors, by the 1960s, many investigators had concluded that the virus was not immunogenic and were not using immunodetection. Further, almost no morphological or cytological differences could be observed between mammary tumor cells and normal cells in tissue culture. MMTV-infected mouse mammary tumor cells are contact-inhibited in vitro and do not form foci, colonies, or plaques as observed in cultures with the transforming RNA and DNA tumor viruses (Vacquier and Cardiff, 1979). Therefore, the simple tissue culture technologies popularized by the new breed of cellular oncologist did not prove applicable to the mammary tumors and their viruses.

The immunology of the virus was soon worked out by Phyllis Blair and others using, at first, the Ouchterlony agar diffusion plates and then increasingly sophisticated immunological techniques (Blair, 1968). These confirmed Andervont's earlier observations of tumor immunogenicity (Andervont and Bryan, 1944) and led the way to other quantitative immu-noassays and opened the way to immunodetection and quantification of the virus (Cardiff et al., 1968).

Experiments at the Cancer Research Genetics Laboratory (CRGL) of the University of California, Berkeley showed that the virus could be produced in large quantities from mammary tumor cells grown at high density and maintained as dome cultures (McGrath, 1971; McGrath et al., 1972; Young etal., 1976). Eventually, cell lines with sufficient virus production (Parks etal., 1974b; Yagi et al., 1978) were established and became a valuable source of MMTV. Several groups developed immunofluorescence for semiquantitative in vitro infectivity assays but even these proved too cumbersome for extensive use (Vacheron et al., 1997; Vacquier and Cardiff, 1979).

With the development of quantitative radioimmunoassays and reverse transcription assays, the virus could be detected readily (Cardiff, 1973; Howk et al., 1973; Lo Gerfo et al., 1974; Parks et al., 1974a; Ritzi et al., 1976; Sheffield et al., 1977; Verstraeten et al., 1973, 1975). These assays provided rapid detection methods but to this day, the field has no effective in vitro assays for the virus.

Through all of this period, the traditionalists resisted the viral concept. In 1968, the eminent virologist Harry Rubin taught his University of

California, Berkeley tumor virology classes that MMTV was not a real virus or, at least, was not tumorigenic.5 Rubin's experience was based on the plaque assays he used so effectively for the study of RSV. The Duesberg and Blair paper (Duesberg and Blair, 1966) proving that MMTV, like the mouse and avian leukemia and sarcoma viruses, had an RNA genome was required to convince a skeptical Dr. Rubin. A further nail was put into that coffin with Duesberg's description of MMTV's bimolecular genome, a feature common to all RNA tumor viruses (Duesberg and Cardiff, 1968). However, not everyone got the message. As late as 1978 papers describing in vitro assays for MMTV were being rejected by eminent virologists because MMTV was "not a transforming virus and probably just a helper virus."5 Much of the uncertainty could have been resolved with the development of a decent bioassay for MMTV.

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