An electron microscope study of virus-like particles in chick embryo and L cell cultures.

Latent virus infections in chickens have been recognized for several years. Many investigators have seen spherical, virus-like particles in normal chicken cell cultures, and have suggested that they might have been the virus of avian lymphomatosis, which occurs frequently without producing disease symptoms and is transmitted trans-ovarially. Virus-like particles were also observed during the present study of the ultrastructure of primary monolayers from Io-day-old chick embryos cultured in Eagle's (I955) medium supplemented with 3 % calf serum. Cultures were prepared according to the method of Dulbecco & Vogt (I954) from a Heisdorf and Nelson hybrid White Leghorn commercial breed of chickens; 56% of the cells had virus-like particles either at their periphery or internally. P1. I, fig. I shows a group of extracellular particles in a chick embryo culture incubated for 4 days; their mean diameter was IO6O ~,(Table I). Each particle consisted of a dense central nucleoid surrounded by a membrane, then a clear area finally bounded by an outer membrane. Both the chicken and murine viruslike particles (discussed later) were fixed with either OsO 4 or glutaraldehyde followed by OsO4 post-fixation, and then studied with a Siemens Elmiskop I electron microscope. Sections were cut on a Servall MTI Porter-Blum ultramicrotome. The particles were almost exclusively extracellular (P1. I, fig. 2). Occasionally they were seen in cytoplasmic vacuoles near the cell surface, and, rarely, in cytoplasmic vacuoles near the nucleus (P1. I, fig. 3). When chick embryo cultures were incubated for 4 days before fixation instead of one, the particles present appeared to have increased greatly in number, although cytopathic effects did not occur. In the chick cells, the production of three different strains of Western equine encephalomyelitis (WEE) virus was not inhibited by the particles. For example, titration data from one strain gave a total WEE virus titre of 4-2 × IO 9 p.f.u./ml, for I3 hr after infection. The electron-microscopic observation of WEE virus multiplying to high titres in chick embryo cells containing these virus-like particles has not been previously reported. Experiments with WEE virus were not done using chick cells or L cells devoid of virus-like particles. However, Morgan, Howe & Rose (I96I) studied WEE virus development using chick embryo cells in which no virus-like particles were observed. In PI. I, fig. 4, WEE virus and virus-like particles are seen together extracellularly, WEE virus having a mean diameter of 480 + 3"3 X (n = 99 WEE particles). Febvre & Benedetti 0958) examined normal White Leghorn, as well as Brown Leghorn, cells and saw virus-like particles only in the former; their Brown Leghorns were supposed to be free from avian lymphomatosis virus. Their particles were similar in morphology to those described here, most of the particles were extracellular, and no cytopathic effects were observed. In the second part of this study, a morphologically different type of virus-like particle was seen in two lines of cultured strain L 929 mouse cells. Lockart (I963) has