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A quantitative ultrastructural study of the development of bluetongue virus in Madin-Darby bovine kidney cells.

Summary
Samples of Madin-Darby bovine kidney cells were taken for electron microscopy at various times after infection with bluetongue virus. A quantitative electron microscopic cell sampling technique was used in observing ultrathin sections of the cell population, and various morphological features were tabulated. The appearance of virus within phagocytic vesicles and lysosomes, and the distension of the rough endoplasmic reticulum, were early ultrastructural changes resulting from the virus infection. Granular inclusions which were usually juxtanuclear and tubular structures were other features observed later in cells infected with bluetongue virus. Progeny virus particles were seen within granular inclusions, amidst tubular structures, and within cytoplasmic vesicles. There was agreement between the infectivity data from plaque counts of cell-associated virus and direct electron microscopic counts of cells containing intracellular virus, granular inclusions, and cytoplasmic tubular structures. The large tubules associated with bluetongue infection had a mean outer diameter of 47.2 nm., more than twice the size of classic cellular microtubules. The mean diameter of bluetongue virus measured in ultrathin sections and negatively stained preparations was 63 nm. Even at late stages of infection, small numbers of intracellular virus particles were observed with the electron microscope. This agreed with the low titres obtained from plaque counts of cell-associated virus. Bluetongue virus did not, therefore, accumulate in the cell and undergo a burst-like release. Instead, it appeared to be extruded from the cell as it was made. There was no evidence for virus release by budding from the plasma membrane, nor for the presence of an envelope around complete particles.