Bovine parainfluenza type 3 virus infection: virus replication in bovine embryonic cell cultures and virion separation by rate-zonal centrifugation

Abstract

Replicative sequences of a bovine strain of parainfluenza type 3 virus in bovine embryonic kidney and spleen cell cultures were investigated by light and fluorescence microscopy and by ultrathin section and negative-contrast electron microscopy. Observations from light and fluorescence microscopy showed that intracytoplasmic inclusions were detected as small granules surrounding the nuclei of more than 90 percent of the cell population by day 2 postinoculation. With the increase of postexposure times, these inclusions coalesced into larger bodies which occupied large portions of the cell. Ultrastructurally, the first sign of virus development was the appearance of aggregates of viral nucleocapsids in the vicinity of the nucleus. With the concomitant accumulation of viral nucleocapsids in the cytoplasm, the virus maturation was expressed by budding processes through the cell membrane into round, oval, or elongated forms. Eosinophilic inclusions were demonstrable in many mitotic cells. Ultrastructurally, these cells were observed to produce virus particles by a process identical to that of resting cells. Virions, prepared from infected culture fluid and negatively stained, appeared to be pleomorphic and their diameter ranged from 200 to 600 mm. The virions were separated, by rate-zonal centrifugation, into two subclasses in a sucrose gradient (15 to 60 percent, wt/wt). The slowly sedimenting virions had a density approximately 1.20 gm/cm3 and an average size of 200 nm in diameter, whereas the faster-sedimenting virions had a density of 1.24 gm/cm3 and average diameter of 400 nm.