Electron microscopic study of the H.R. strain of herpes simplex virus in cells of the chicken chorioallantoic membrane (1) suggested that the virus differentiates within the nucleus, where particles possessing a central core and a single limiting membrane are randomly dispersed. In this earlier work the finding that ruptured nuclei were associated with large numbers of intracytoplasmic particles possessing double membranes appeared to be consistent with the hypothesis that the virus acquired a second membrane in the cytoplasm after release from the nucleus. However, cells in tissue culture infected with the H F E M strain of herpes simplex virus and examined by Stoker, Smith, and Ross (2) and herpes B virus examined by Reissig and Melnick (3) subsequently revealed intranuclear particles with double as well as single membranes, thus raising the possibility that development could be completed in the nucleus.Recently, a strain (J.M.) of herpes simplex virus was found to crystallize (4) in a manner analogous to the adenoviruses (5-9). Moreover, the nuclear membranes of cells infected by this strain frequently showed remarkable proliferation. The purposes of this paper are to illustrate and describe the viral crystals as well as the morphology of the cellular response to infection, and to propose an hypothesis concerning the manner of development and the mechanisms whereby virus may gain egress from intact cells. Materials and MetkodsHeLa cells were cultured in a medium consisting of Earle's balanced salt solution which contained 0.5 per cent lactalbumin hydrolysate, 0.25 per cent glucose, 0.1 per cent yeast extract, 0.3 per cent tris(hydroxymethyl)-aminomethane and 20 per cent horse serum. Stable human amnionic cells were cultivated in Eagle's medium to which 20 per cent horse serum * These studies were

Western equine encephalomyelitis (WEE) virus is 40 to 55 m/z in diameter (1) and apparently contains nucleic acid of the ribose type (2, 3). Although electron microscopic examination of whole mounts of tissue cultures infected with a closely related agent (Eastern equine encephalomyelitis virus) has shown viral particles both within and on the surface of cells (4,5), no studies employing thin sections have been previously reported. The purpose of this communication is to illustrate and describe the manner in which WEE virus appears to differentiate within, and gain egress from, infected tissue culture cells as revealed in thin sections by the electron microscope. Similarities in development or release among WEE virus, influenza virus, herpes simplex virus, the virus associated with erythroblastosis of chickens, Rous sarcoma virus and a mouse mammary tumor agent will be discussed. Stable human amnionic cells:A stable line of human anauionic epithelium was kindly supplied by Dr. Katherine Sprnnt. Replicate cultures were prepared in Leighton tubes without coverslip% using Eagle's basal medium with 10 per cent horse serum. The cultures were inocu-* These studies were aided

Electron microscopic studies of influenza virus have been reported periodically for more than a decade, but the knowledge thus gained concerning the structure and especially the development of this group of viral agents is still rudimentary.Early work by Taylor, Sharp, and associates (I-3) disclosed that strains of swine, type A (PRS) and type B (Lee) virus, isolated by centrifugation from the chorioallantoic fluid of infected chicken embryos, were composed of sphcrical or ovoid particulate units, with average diameters of approximately 78, 78, and 97 m/~, respectively. These investigators also observed that the viral particles apparently possessed an "internal differentiation in the structure, marked by a single region of relatively high density in the individual particles." Mosley and Wyckoff (4) first described elongated or filamentous forms in preparations of the PRS, Weiss, and Lcc strains of virus, noted that these rod-like structures frequently appeared to bc partly segmented into spherical particles having the same dmmeter, and suggested that there was a significant relationship between the two forms. It has since been shown that both filamentous and spherical forms can usually be demonstrated in preparations of influenza virus from infected chorioaUantoic fluids, regardless of the strain employed, although filaments are espccially numerous in recently isolated A strains (5). Moreover, filaments have been seen in tissue cultures of infected chorioallantoic membrane (6), as wcU as in thin sections of infected membrane and mouse lung (7-9). In connection with these latter observations, it has been pointed out that the viral particles appear to develop solely from the surface of cells and that the filaments often show segmcntation into spheres. Consequently, it is scarcely surprising that "the most general interpretation is that the filaments represent an intermediate stage in the multiplication of virus and that the viral par-

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