M. A. Epstein

Cells of the Raji strain, established in vitro in Nigeria from a Burkitt lymphoma, were maintained and studied between the 16th and 28th months of culture. As judged by their mode of growth in suspension, appearance when stained, and fine structural organization, the cells were undifferentiated lymphoblasts similar to those of other tissue culture strains from Burkitt tumors. Characteristics suggesting a lack of differentiation were the formation of large aggregations containing many hundreds of individuals, a relatively large diameter, irregularly indented nuclei, and a fairly extensive cytoplasm with free ribosomes tending to clump. No virus particles could be found in the cells by electron microscopy, despite intensive searching of 16 separate samples. Although the cells were resistant to vesicular stomatitis virus, this resistance could not be transferred to other normally susceptible test cultures and an interferon-like inhibitor could not be found. The Raji cells could, however, produce interferon when stimulated by Newcastle disease virus. Unlike other cultured Burkitt tumor cells, Raji cells did not respond positively in immunofluorescence tests. The failure to detect virus particles or indirect evidence of virus infection in Raji cells is discussed.

A panel of 24 healthy seropositive donors have been followed prospectively over a period of 15 months and monitored (1) for the level of EB virus shedding in the throat by means of a sensitive cord-blood transformation assay; (2) for the level of virus-infected B cells in the blood via a new in vitro protocol where "spontaneous transformation" can be seen to titrate against input cell number; (3) for anti-EB viral antibody titres and (4) for the prevailing level of virus-specific memory T cells in the circulation. Six donors shed easily detectable levels of EB virus into throat washings on every occasion of testing, whilst 16 other donors shed lower levels of virus detectable in throat washings on a majority (10 donors) or on a minority (6 donors) of test occasions; only 2/24 donors gave no evidence of virus shedding at any time. There was a direct relationship between the EB virus shedder status of an individual (i.e., the level of virus replication in the pharynx) and the number of infected B cells present in the circulation. These results indicate that chronic, usually low-grade, replication of the virus at some permissive site in the oro- and/or naso-pharynx is very often a stable accompaniment of the asymptomatic EB virus carrier state, and may indeed be essential for the long-term maintenance of that state.

Lymphoblasts of two tissue culture strains (EB1 and EB2) from different biopsy specimens of Burkitt's lymphoma have been examined in thin sections by electron microscopy, and have each been found to carry a morphologically identical virus. The virus was observed in samples taken over many months, being present in about 1 to 2 per cent of the cells in two forms: Immature particles about 75 mmicro in diameter which were seen in both the nucleus and cytoplasm; and larger mature particles with a diameter of 110 to 115 mmicro, which were either within membrane-bounded cytoplasmic spaces or at the cell surface. There was some indication that the particles matured by budding through the cytoplasmic membranes. Both types of particle occurred in dead degenerating cells or, less frequently, in intact altered cells. The characteristic alterations of the latter included margination of the chromatin, fragmentation of the nuclear envelope, beaded opaque material in the mitochondria, and, with one of the cell strains (EB1), sheaves of altered spindle tubules. All attempts to isolate and identify the virus carried by the two strains of lymphoblasts failed. No pathological effects were caused in 8-day chick embryos inoculated either with whole lymphoblasts or extracts of disrupted lymphoblasts, using the intraallantoic, amniotic, and chorioallantoic routes, and the extraembryonic fluids of such chicks were without haemagglutinating activity for human, chicken, guinea pig, or monkey erythrocytes. Whole lymphoblasts or lymphoblast extracts were likewise without effect when inoculated intraperitoneally into newborn hamsters or two strains of newborn mice. Similar lymphoblast inocula did not cause detectable changes in 9 different test tissue culture systems even after 8 blind passages. The nature of the unknown, unidentified virus in the cultured lymphoblasts from Burkitt's lymphomas is considered and its possible relationship to the cells discussed.