Virus of Avian Erythroblastosis. VII. Ultra-structure of Erythroblasts From the Chicken and From Tissue Culture2

Abstract

Studies have been made on culture in vitro of the erythroblasts occurring in the circulating blood of chickens with erythroblastosis. In 23 experiments during the past 3 years, growth of the cells was observed for more than 80 days in 3 of the studies. Cell proliferation has been dependent on a culture medium consisting of 50 percent normal chicken serum in medium 199 which contained, in the more recent experiments, vitamins in the concentration found in Eagle's medium. Glucose was added to medium 199 in a concentration of 0.5 percent. The average generation time measured in 1 experiment was 9.9 days. Concurrent with maintenance and growth of the cells in culture, erythroblastosis virus was liberated continually for periods up to 125 days, as demonstrated by electron micrography of thin sections. The observation of typical virus particles in the culture fluids was corroborated by the induction of the characteristic erythroblastosis in susceptible chickens. An investigation was made also of the ultrastructure of erythroblasts and other cells in the diseased bird and of the erythroblasts after various periods in tissue culture. Numerous cells of the character of macrophages, and reticular and endothelial cells were seen in the bone marrow and spleen which contained large numbers of virus particles in vacuole-like structures. Virus was seldom observed in the erythroblasts in the tissues or in the circulating blood. Most of the virus in the erythroblasts under these conditions was collected in vacuoles, but an occasional virus “bud” occurred in the cell membranes. Erythroblasts in the circulating blood exhibited increased vacuolization. The culture of erythroblasts resulted in little, if any, increase in virus associated with the cell. Some cells contained collections of virus in vacuoles, which had the appearance of originating by the process of phagocytosis of the agent seen in considerable amounts outside the cell. The probability of the occurrence of phagocytosis was supported by the demonstration that the erythroblasts in the cultures ingested large numbers of India ink particles, among which virus particles were seen in the vacuoles. Buds, which had the appearance of formation of virus particles, were seen frequently in the cell membrane. The particles in the process of formation contained no nucleoids. The findings were interpreted to indicate synthesis of the virus in immature form in the cell membrane and maturation of the agent with formation of the nucleoid outside the cell. No evidence was found to indicate the synthesis of virus in mitochondria or any other cellular structure. The results were discussed in relation to findings with myeloblasts in which the specific virus is synthesized in walled bodies inside the cell by processes not involving the cell membrane.