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Chorioallantoic Membrane URSULA FELLER, ROBERT M JOURNAL OF VIROLOGY, Nov. 1969, p. 753-762 Vol. 4, No. 5 Copyright © 1969 American Society for Microbiology Printed in U.S.A. Morphogenesis of Newcastle Disease Virus in Chorioallantoic Membrane URSULA FELLER, ROBERT M. DOUGHERTY, AND HENRY S. Di STEFANO Departments of Anatomy and Microbiology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 Received for publication 2 July 1969 Chick embryo chorioallantoic membrane, infected with the Blacksburg strain of Newcastle disease virus, was examined with an electron microscope to investigate the sequence of viral-induced host cell alterations. These were evident mostly in the endodermal epithelial cells lining the allantoic sac and were divided arbitrarily into three stages. Stage 1 was characterized by commencement of cell hypertrophy and hyperplasia and presence of fewer cytoplasmic inclusion bodies normally found in the cells; in stage 2, juxtanuclear nucleocapsid-glycogen aggregates appeared, and there were increased numbers of microvilli; stage 3 was characterized by increased cytoplasmic density and evidence of viral assembly and release. The morphological features of viral assembly and the virion are also described. Most morphological descriptions of Newcastle RESULTS disease virus (NDV) have been based on electron The Blacksburg strain of NDV was chosen for microscopy studies utilizing negative staining these studies, because it is attenuated virus which techniques (8, 13, 23, 24). There have been no permits prolonged survival of detailed descriptions of the viral fine structure infected cells, in an in material the attempt to determine an orderly sequence of and assembly sectioned since early events. At 10 days of development, chick em- work of Bang (1), who reported evidence of viral bryos were infected with 106 ID50 of the Blacks- release from a modified cell surface and some burg strain of NDV by injection into the allan- alterations in endodermal epithelial cells of chick toic and embryo chorioallantoic membrane (CAM) after sac, infected and uninfected control NDV. embryos were incubated at 37 C. Infected and infection with The purpose of this report control eggs were removed at various intervals is to extend the earlier observations and to de- after injection (16, 25, 48, 72, 90, 95, and 114 hr), scribe some of the finer details of cellular altera- and pieces of CAM were processed for electron tions and viral morphogenesis made possible by microscopic examination. more refined modern preparative techniques of The allantoic sac of 10-day chick embryos is electron microscopy and the improved resolution lined with approximately 108 cells (12). Each egg and magnifications available today. was infected at this age with 106 1D50 of virus, MATERIALS AND METHODS therefore infection was asynchronous. Thus, at each time interval, cells were evident at various The Blacksburg strain of Newcastle disease virus was generously supplied by Vincent Groupe. Stock stages in the viral growth cycle. Under these virus was prepared by injection of 10-day chick conditions, the exact sequence of events could not embryos in the allantoic sac with approximately 106 be determined. However, trends in the overall 50% infectious doses (1150) of virus. Infected allan- sequence were obvious and the morphological toic fluid was harvested after 48 hr at 37 C and stored events after infection were divided arbitrarily at -65 C. Stock virus contained 108 ID60/ml. For into three stages. electron microscopy, samples of CAM were fixed Cell alterations. The most obvious morpho- overnight at 2 C in a phosphate-buffered modified logical alterations resulting from infection of the Bouin fixative (26). The tissues were washed subse- allantois were hyperplasia of the epithelial cells, quently with the phosphate buffer, treated with a 1% as solution of osmic acid in phosphate buffer for I hr, described earlier by Bang (1, 2), and changes washed again with the buffer, dehydrated, and em- in their cytoplasmic composition. The latter were bedded in Maraglas (10). Thin sections were stained particularly manifested in the layer of epithelial with uranyl acetate (25) followed by basic lead citrate cells lining the allantoic sac. The fine structure of (22) and examined with an electron microscope endodermal cells of the CAM has been described (Norelco EM 300) at an accelerating voltage of 80 kv. in detail by Borysko and Bang (6) and Leeson 753 754 FELLER, DOUGHERTY, AND Di STEFANO J. VIROL. and Leeson (16). The uninfected allantoic mem- maximum of two cell layers to four or five cell brane consists of one or two layers of flattened layers. The most obvious cytoplasmic alterations epithelial cells containing the usual cytoplasmic at this stage were noted in cells lining the allan- organelles (Fig. 1). The luminal surface of these toic cavity. In some cells there was a marked cells is relatively smooth with few microvilli. A increase in the number of microvilli extending prominent feature of these cells at 10 days of into the allantoic sac (Fig. 2), as reported previ- incubation is the presence of dense, membrane- ously (1, 3). The characteristic electron-dense bounded inclusions composed of finely packed granular inclusions were fewer and less dense, granular material. and contained loosely packed granular sub- The earliest viral-induced alterations (stage 1) stance (Fig. 3). in the allantoic membrane were seen 48 hr after The second phase (stage 2) was characterized infection; among these were epithelial hyper- by the consistent appearance in the vicinity of trophy and hyperplasia. By 114 hr after infection, the nucleus of large accumulations of particles the epithelial lining increased from a control (Fig. 4) with the morphology and staining charac- teristics of glycogen (21). These particles meas- ured 15 nm in diameter and stained faintly with uranium salts and heavily with lead. Similar particles were seen randomly scattered in the cytoplasm of comparable control cells, but not in the concentrated clusters characteristic of in- fected cells. At about the same time, also in the vicinity of the nucleus, aggregates of filamentous structures were observed (Fig. 4). These measured 10 to 13 nm in diameter, and had transverse striations with a periodicity of 5 to 5.5 nm (Fig. 5). They are similar in appearance to the threadlike struc- tures and filaments that have been described in tissue culture cells infected with viruses of the mumps-NDV-parainfluenza group and which are believed to be the viral nucleocapsids (5, 17, 18). In many cases, the filamentous aggregates were found in close association with large clus- ters of glycogen granules, and frequently mito- chondria were seen immediately adjacent to this glycogen-filament complex (Fig. 4). The most significant feature of stage 3, which was observed 72 hr after infection, was viral assembly and release (Fig. 6). Cells which were actively producing and releasing viral particles showed greatly increased cytoplasmic density due to the presence of large numbers of free ribosomes and diffuse accumulations of the fila- mentous material described above, which we believe to be the viral nucleocapsid. Whereas earlier this filamentous material was in juxta- nuclear aggregates, it now appeared mostly con- centrated subjacent to the luminal surface. The glycogen granules that previously were clustered and in close association with the juxtanuclear filamentous aggregates were now scattered within the cytoplasm. At this stage, the microvilli appeared still more numerous and more promi- FIG. 1. Endodermal cells of allantoic membrane nent than in the previous stage, and they were from 15-day-old control chick embryo. Note scarcity of longer and frequently forked or branched. microvilli and presence of electron-dense granular in- Viral assembly. Viral subunits were assembled clusions (i) in cell lining the allan2toic cavity (AC). either as filaments or spheres. In each case the X11,200. morphogenesis of NDV resembled that described VOL. 4, 1969 MORPHOGENESIS OF NDV IN CAM 755 AC 2 FIG. 2. Epithelial cells of allantois from 12-day-old chick embryo 48 hr after infection with the Blacksburg strain of ND V. In cell layer lining allantoic cavity (AC), note proliferation of microvilli antd hypertrophy in cell at left, and unaltered appearance of cell at right. X 6,400. 3 .. e~~ 4. ^.,O _ I - -.s.. 4 is.0 0f- %v i X -A l A . s5 .8 FIG. 3. Part of endodermal cell lining allantoic sac from 14-day-old chick embryo 95 hr after inifection with ND V. Several granular cytoplasmic inclusion bodies of low electron density (arrowheads) are visible below the cell surface (compare Fig. 1). X27,900. _w :00,I 4 Ol2 0%X '^o ^ tK gl* .4. I - *.A lhb.l v FIG. 4. Endodermal cells lining the allantois of 15-day-old chick embryo 114 hr after infectionz with ND V. Aggregates offilamentous structures (F) and accumulations ofglycogen particles (G) are in cytoplasm niear nzucleus (N). A similar aggregate in adjacent cell is at upper left. Allantoic cavity is to right. X 15,400. Filamentous nature ofaggregates and some glycogen particles are shown in inset, which is higher magnification of area A. )X 37,650. 756 VOL. 4, 1969 MORPHOGENESIS OF NDV IN CAM 757 FIG. 5. High magnification ofa filamentous aggregate in endodermal cell lining allantoic cavity from 14-day-old chiick embryo 95 hr after infection with ND V. Note periodic crossbanding offilaments (arrows). X 119,700. with parainfluenza viruses (4, 7). Typical budding 100 nm along their entire length. Frequently forms of the Blacksburg strain of NDV charac- they showed the presence of a clublike ending. teristic of the filamentous and spherical types are Most morphological descriptions of NDV have shown in Fig. 7, 8, and 9. The viral envelope been based on studies of negatively stained prep- was derived from, and therefore continuous with, arations (8, 13, 23, 24). These indicated that the the host cell plasmalemma.
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