ISOLATION OF RETROVIRUS PARTICLES IS AN ABSOLUTE PREREQUISITE FOR THE IDENTIFICATION OF RETROROVIRAL "MARKERS"

Etienne de Harven* and Gordon T. Stewart**

Abstract

In the early studies on RNA tumor viruses, now classified as "retroviruses,i, associated with several forms of leukemias and cancers in mice and chickens, isolation of the viral particles was regarded as the essential preliminary step for significant biochemical analysis. Isolation implies separation of the viruses from everything else, therefore obtaining a pure preparation of viruses. The success of these isolation procedures was systematically monitored by electron microscopy of the virus particles pelletized by high speed ultracentrifugation and prepared by the thin section methodology. Such preparations provided adequate samples for reliable identification of enzymes, nucleic acids and proteins of murine and avian neoplasia associated viruses, with minimal risk of confusion with cellular debris.

In sucrose density gradients, "retroviruses" sediment around 1.16gm/ml. This property was used, in the 1970s, in many attempts to isolate retroviruses from human leukemias and cancers, as well as in further studies of avian and murine RNA viruses. It was known, however, that many microvesicles and cellular debris also sediment at the density of 1.16gm/ml. Strict controls by electron microscopy were therefore absolutely necessary before considering that the 1.16 bands represented "pure virusn. Omitting this control was opening the door to dangerous misinterpretations, i.e. regarding proteins, RNA fragments, or enzyme activity as belonging to the virus while it could have been just as well originating from contaminating cellular debris and microvesicles. In the historic papers which appeared in 1970 and in which a reverse transcriptase (RT) activity was claimed as that of a viral enzyme, electron microscopy controls were ignored and the enzyme activity could have been just as well of cellular, non-viral origin.

Intensive and highly funded efforts to demonstrate a hypothetical association between "retroviruses" and human cancer having failed in the 1970s, most of the involved retroviral oncologists jumped in the early 1980s on the apparent ~epidemic" of AIDS in the hope of salvaging the research interest of "retroviruses" by focussing on HIV. Pathetically, methodolegy was still primarily based on the false assumpbon that sucrose 1.16 bands represented "pure retroviruses", omitting the essentia! electron microscopy controls. Several proteins, enzymes and RNA fragments were quickly idenmied in these samples and pronounced as "retroviral markersn. Elisa and Western Blot tests which have been used ever since for the diagnosis of the so-called "seropositivityn and of infection with live HIV were entirely based on the use of such "markers".

To those who knew how inadequate the methodology had been, it was no surprise to learn, in 1993, from the extensive investigations conducted by the Australian group in Perth (1) that the HIV antibody testing was non-specific. It took 15 years for the AIDS research establishment to finally look, by electron microscopy, at the 1.16 bands of supposedly HIV producing cell lines and to find out that these bands contained massive amounts of cell debris, and barely few virus-like particles...(2,3). Publications 1,2,and 3 have been conveniently ignored by the AIDS orthodoxy, worldwide.

1) Papadopulos et al., Bio/Technology 11:696-707, 1993; 2. Glushanhof et al., Virology 230:125-133, 1997; Bess et ai., Vrology 230:134144, 1997.

*Emeritus Professor of Pathology, University of Toronto, Canada.

** Emeritus Professor of Public Health, University of Glasgow, UK.