epithelial cells that are seen following infection of infant mice with have firmly established its viral etiology. The virus is a

Transcription

1 ELECTRON-MICROSCOPIC STUDY OF THE INTESTINAL EPITHELIUM OF MICE INFECTED WITH THE AGENT OF EPIZOOTIC DIARRHEA OF INFANT MICE (EDIM VIRUS) W. ROBERT ADAMS, M.D., AND LISBETHI M. KRAFr, D.V.M.* From the Veterans Administration Hospital, West Haven, Conn., and the Department of Pathology, Yale University School of Medicine, New Haven, Conn. The highly contagious virus disease of infant mice that has recently come to be known as epizootic diarrhea of infant mice (EDIM) was first described in I947 by Cheever and Mueller.' These authors were persuaded that the disease was infectious in nature and probably had a viral etiology, but did not believe that their data furnished sufficient evidence to prove these points. The epidemiology and pathology of EDIM has now been reported in some detail,1-6 and transmission experiments7 have firmly established its viral etiology. The virus is a heat-resistant particle that can be serially transferred and is neutralized by specific hyperimmune antiserum from rabbits.7'8 Intracytoplasmic virus-like particles interpreted as EDIM virus have been recently demonstrated to occur in close association with the endoplasmic reticulum of intestinal epithelial cells of infected mice.9 The electron-microscopic studies described in this report were designed to characterize the ultrastructural changes in the intestinal epithelial cells that are seen following infection of infant mice with EDIM virus. MATERIALS AND METHODS Method of Infection Infant mice of the ICR strain, I-3 days old, were infected by oral administration of IO5-IOS ID50 of EDIM virus in the form of a purified infective intestinal filtrate prepared as previously described.7 The mice were from a colony shown to be free of and susceptible to EDIM virus infection.8 Fixation and Embedding Approximately i day after the appearance of overt symptoms of diarrhea, the mice were killed by decapitation, the small intestine immediately exposed, and portions of the jejunum fixed in i% osmium tetroxide buffered to ph 7.2 in o.o6 M veronal-acetate buffer for i-4 hr. The specimens were then dehydrated in graded Supported by Research Grants CA-o2738, from the National Cancer Institute, and AI-o6584 and AI-o4374 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. * Present address: Oak Ridge Institute for Nuclear Studies, Oak Ridge, Tenn. Accepted for publication Jan. 23, I

2 40 ADAMS AND KRAFT Vol. Sz, No. r alcohols and embedded in methacrylate. Sections were cut with a glass or diamond knife on a Sorvall Porter-Blum type microtome, stained with lead hydroxide10 or uranyl acetate-lead citrate1' and examined in an RCA EMU 3G or Siemens Elmiskop i electron microscope. Material for light microscopy was fixed in Bouin's fixative, or in Zenker's fixative with or without acetic acid, and embedded in paraffin; routine sections were stained with hematoxylin and eosin, Masson's trichrome stain, Laidlaw's inclusion-body stain,'2 alcian green stain for mucin, or Giemsa stain. RESULTS Light Microscopy One of the distinguishing features of this disease was the total lack of a cellular inflammatory exudate, even in severely affected portions of the bowel (Fig. i-8). The morphologic changes appeared to be limited exclusively to the extreme tips of the villi, which became bulbous as a result both of congestion of the small blood vessels and dilatation, in some cases extreme, of the lymphatics. Changes in the epithelial cells were limited to those covering the tips of the villi. The least severe changes consisted of a fine vacuolation affecting the cytoplasm of the cell between the nucleus and the striate border. This vacuolation had a fine vesicular appearance in the least affected cells, whereas in those most severely affected, large empty vesicles occupied the entire cytoplasm with concomitant disappearance of the usual complement of cytoplasmic organelles and matrix. The striate border remained intact in cells with mild and moderate stages of vacuolation, but when the latter was extreme, the border became greatly attenuated and finally disappeared. The nuclei of affected cells were relatively normal in appearance except where cytoplasmic vacuolation was extreme. Under this condition, the nuclei assumed a shrunken and pyknotic appearance. Large quantities of cellular debris could be seen within the lumen of the affected intestine, undoubtedly the result of massive sloughing of the epithelial cells. The extensive fluid losses accompanying the diarrhea that characterizes this disease would appear to result from the extensive epithelial sloughing at the dilated edematous tips of the villi. Cytoplasmic acidophilic inclusion bodies similar to those described by Pappenheimer 4 were seen (Fig. 6-8). These inclusions were usually circular or slightly ellipsoidal in cross section and measured from i to 4 1 in their greatest dimension. They were demonstrable with most of the stains employed but were most easily visualized in sections stained with Laidlaw's inclusion-body stain.'2 There appeared to be no preferential position for these cytoplasmic inclusions, approximately equal numbers being found in the juxtanuclear (Fig. 6), supranuclear (Fig. 7), and infranuclear (Fig. 8) positions. Intranuclear inclusions were never seen.

3 July I967 EDIM INFECTION 41 Electron Microscopy The least severe changes noted (Fig. 9) consisted of simple dilation of both smooth- and rough-surfaced endoplasmic reticulum. These dilated sacs of reticulum always contained varying quantities of a stippled, finely granular precipitate. Accompanying the dilatation were accumulations of lipid material within the sacs of reticulum (Fig. 9-I1). When the dilatation of endoplasmic reticulum and the accompanying deposition of lipid was rather severe, recognizable virus particles could be seen within the dilated sacs (Fig. 9-II and I3). The nucleus, mitochondria, and other cytoplasmic organelles of affected cells showed little if any morphologic change except when the cell had been converted to a virtual bag of lipid with virus particles sequestered in contiguous hugely dilated sacs of endoplasmic reticulum (Fig. i i). The microvilli remained unchanged in cells minimally or moderately involved (Fig. 9 and io) but were short, thick, and irregular in more severely affected cells (Fig. ii and I2). The Golgi apparatus was never significantly involved, the only notable change being minimal dilation in severely involved cells. Virus was never seen in Golgi vesicles. The material examined at a given time interval after infection contained cells with a spectrum of morphologic change varying from those indistinguishable from normal through a series of affected cells displaying severe morphologic changes. In general, the morphologic spectrum tended to correspond to the spatial distribution of the cells. There appeared to be a gradient of cellular susceptibility to infection, as judged by morphologic response, increasing in sensitivity from crypt to tip of villus (Fig.2 andi2). The majority of virus particles visualized in the infected cells were present within vesicles. These vesicles varied considerably in size, in some cases being as large as or larger than the nucleus of the cell (Fig. IO, ii, and I3), and by virtue of the ribosomes attached to their outer aspect (Fig. I3, I5, I7, and I8) could be identified as elements of the rough-surfaced reticulum. The virus particles were in many cases associated with the membrane of the endoplasmic reticulum in such a way as to suggest formation in the cytoplasm outside the endoplasmic reticulum (Fig. 21). In the electron micrographs particles appeared to protrude into the lumen of the endoplasmic reticulum, becoming invested in the process by an external coat apparently derived from the endoplasmic reticulum membrane (Fig. I4-2 i). A somewhat puzzling feature was the lack of such an external envelope on some of the intravesicular particles (Fig. 20 and 2I). This suggested that entrance into the vesicle of endoplasmic reticu-

4 42 ADAMS AND KRAFT Vol. SI, No. z lum could be gained without such investiture or that the membranederived outer envelope of the virus particle was variable or transient in nature. The distribution of virus-containing vesicles within the cytoplasm of infected cells was uniform throughout the cell. They were seen in the infranuclear, paranuclear, and supranuclear regions with approximately equal freuency. The acidophilic inclusion bodies seen in the light microscope could not be identified with certainty in the electron micrographs and hence could not be related to any ultrastructural component of infected cells. It is pertinent that the frequency of these acidophilic inclusions as judged from the paraffin sections was considerably less than the frequency of virus-containing cells as judged from the electron micrographs. This strongly suggests that, whatever their nature, the inclusions were not merely virus-filled vesicles. A number of electron micrographs, one of which is shown in Fig. I2, indicated that liberation of virus particles into the intestinal lumen occurred by way of rupture of afflicted cells with the disgorgement of both ruptured and intact virus-containing vesicles as well as large quantities of the virus-associated lipid into the intestinal lumen. A summary of the manifold morphologic variations, both of particle morphology and of the particles' relationships to the rough-surfaced reticulum, is presented in Fig. I3 through 22. The general particle form (Type i) averaged 8o mt in diameter and was circular in profile, suggesting that the particles were in most cases spheres; however, rare elongated or ellipsoidal forms were noted (Fig. i9). A typical round particle (Fig. I4) was composed of a circular, centrally located, dense core or nucleoid surrounded in turn by 2 sets of double membranes, the outer set of which could be seen to be in continuity with the vesicle membrane in the case of those particles that were protruding into the vesicle or were still attached to the membrane by a pedicle (Fig. I4-I 7, and I9). Most particles were present within vesicles of rough-surfaced reticulum, but occasional condensations of dense granular material occurred within the cytoplasm proper in which could be seen the faint outlines of round virus particles, possibly in early stages of formation (Fig. 2I). Other studies have indicated that similar cytoplasmic aggregates are nascent viroplasm, and the present material gave no indication that another interpretation is necessary here. In some cases virus particles were identified within dilated vesicles of endoplasmic reticulum that were in direct continuity with the nuclear envelope (Fig. i8); however, particles were never seen within the nucleus proper. A second class of particles (Type 2), averaging 65 mfa in diameter,

5 July I967 EDIM INFECTION 43 was occasionally seen (Fig. I7 and 20). These were similar to the first particle type except for their lack of the outer double membrane. Whether these particles had become divested of this outer coat achieved on passage through the vesicle membrane or whether they had simply failed to acquire such an outer vesicle-derived membrane was not suggested by any of the electron micrographs. A third possibility, of course, is that the latter smaller particles have become sequestered in a vesicle under unfavorable conditions and are either degenerating or have been altered in development in some way. The possibility that they are degenerating is suggested by the fact that in many cases not only do these smaller particles lack an outer membrane, but threads of material, interpreted as the virus nucleoprotein, extend from the core of the virus through apparent morphologic defects in the virus inner shell (Fig. 20 and 22). A third and rare type of elongated or ellipsoidal particle is shown in Fig. I9. It is possible that these are nonviable forms or that they represent developmental aberrations. Virus or virus-associated lipid was never seen in sections of ileum and jejunum from uninfected mice. Such control epithelium was always normal in appearance, resembling the cell at the lower left in Fig. 9. SUMMARY A light- and electron-microscopic study of intestinal epithelium of infant mice infected with epizootic diarrhea of infant mice (EDIM) virus has shown that (i) intracellular virus is seen exclusively in the cytoplasm of infected cells; (2) virus replication is probably initiated in the cytoplasmic matrix, after which the particle appears to enter cisternae of the granular endoplasmic reticulum by a process of "budding;" (3) the endoplasmic reticulum of infected cells appears dilated and contains virus particles and virus-associated lipid; (4) some infected cells (probably the most severely affected) rupture, liberating large numbers of virus particles into the intestinal lumen; and (5) cells exhibit a gradient of susceptibility (as judged by morphologic response) to infection, increasing in sensitivity from crypt to tip of villus. REFERENCES I. CHEEVER, F. S., and MUELLER, J. H. Epidemic diarrheal disease of suckling mice. I. Manifestations, epidemiology, and attempts to transmit the disease. J Exp Med 85: , I PAPPENHEIMER, A. M., and ENDERS, J. F. An epidemic diarrheal disease of suckling mice. II. Inclusions in the intestinal epithelial cells. J Exp Med 85: , 1947.

6 44 ADAMS AND KRAFT Vol. 51, No. z 3. CHEEVER, F. S., and MUELLER, J. H. Epidemic diarrheal disease of suckling mice. III. The effect of strain, litter, and season upon the incidence of the disease. J Exp Med 88:309-3I6, I PAPPENHEIMER, A. M., and CHEEVER, F. S. Epidemic diarrheal disease of suckling mice. IV. Cytoplasmic inclusion bodies in intestinal epithelium in relation to the disease. J Exp Med 88:3I7-324, I RUNNER, M. N., and PALM, J. Factors associated with the incidence of infantile diarrhea in mice. Proc Soc Exp Biol Med 82:I47-I50, I CHEEVER, F. S. Epidemic diarrheal disease of suckling mice. Ann NYAcad Sci 66:I96-203, I KRAFT, L. M. Studies on the etiology and transmission of epidemic diarrhea of infant mice. J Exp Med I06: , I KRAFT, L. M. Observations on the control and natural history of epidemic diarrhea of infant mice (EDIM). Yale J Biol Med 3I:I2I-I37, I ADAMS, W. R., and KRAFT, L. M. Epizootic diarrhea of infant mice: Identification of the etiologic agent. Science 141: , I963. 1O. MILLONIG, G. A modified procedure for lead staining of thin sections. J Biophy Biochem Cytol zz: , I96I. ii. REYNOLDS, E. S. The use of lead citrate at high ph as an electronopaque stain in electron microscopy. J Cell Biol z7:208-2i2, I963. I2. PAPPENHEIMER, A. M., and HAWTHORNE, J. J. Certain cytoplasmic inclusions of liver cells. Amer J Path 12: , I936. I3. MORGAN, C., ROSE, H. M., HOLDEN, M., and JONES, E. P. Electron microscopic observations on the development of herpes simplex virus. J Exp Med zio: , I959. I4. MORGAN, C., ELLISON, S. A., RoSE, H. M., and MooRE, D. H. Structure and development of viruses observed in the electron microscope. II. Vaccinia and fowl pox viruses. J Exp Med 100:30I-308, I954. I5. BERNHARD, W., OBERLING, C., and VIGIER, P. Ultrastructure de virus dans le sarcome de Rous leur rapport avec le cytoplasme des cellules tumorales. Bull Ass Franc Cancer 43: , I956. i6. JENSON, A. B., RABIN, E. R., PHILLIPS, C. A., and MELNICK, J. L. Reovirus encephalitis in newborn mice: An electron microscopic and virus assay study. Amer J Path 47: , I965. The skilled technical assistance of Miss Lillemor Wailmark is gratefully acknowledged.

7 July I967 EDIM INFECTION 45 [ Illustrations follow ]

8 ADAMS AND KRAFT 46 Vol. 5r, No. z LEGENDS FOR FIGURES Figures I to 8 show intestinal epithelium (jejunum) of control and infected mice fixed in Bouin's solution, embedded in paraffin, and stained with Laidlaw's inclusionbody stain. FIG. I. Normal control. Columnar cells are uniform in size and appearance and extend from base of villi to tips. Stroma of microvilli is normal in appearance and there is little if any tendency to club formation of tips. Vacuoles are rare and small. X I8o. FIG. 2. Epithelium from infected mouse showing enlargement and vacuolation of cells over tip of villi, increased cellularity of stroma, and dilatation of stromal lymphatics. Submucosal edema is also present. X i8o. FIG. 3. Tip of villus from normal control showing features mentioned in Fig. i and also demonstrating uniform layer of microvilli and general uniformity and polarity of cells and nuclei. X 360. FIG. 4. Tip of intestinal villus from infected mouse, showing coarse and fine vacuolation and enlargement of cells over tip of villus and dilatation of lymphatics in stromal core. Layer of microvilli is irregular over tip and has disappeared over most severely affected cells. X 360. FIG. S. Portion of cross section of villus from normal control showing uniformity of microvilli, cytoplasm, and nuclei. X I335. FIG. 6 to 8. Cross sections of portions of villi from infected mice showing cytoplasmic inclusions. These may be distinguished from small particles in embedding media by halos surrounding the latter. No intranuclear inclusions were seen. Dark structures suggestive of inclusions, present in some nuclei, are interpreted as normal nucleoli. X 1335.

9 July I967 EDIM INFECTION

10 48 ADAMS AND KRAFT Vol. 51, No. I Figures 9 to 22 are electron micrographs of jejunal epithelium from infected mice. The tissues were fixed in veronal acetate-buffered i% osmium tetroxide and embedded in methacrylate. Sections were poststained with uranyl acetate and lead citrate. FIG. 9. Cross section of villus of infected mouse showing supranuclear and infranuclear vesicles of granular reticulum containing virus particles and irregular clumps of lipid material. Nuclei, cell organelles, and microvilli appear uninvolved. Central cell has both infranuclear and supranuclear vesicles that contain virus particles and lipid. It is flanked by 2 less-involved cells that contain lipid but no visible virus. Cell at extreme lower left is normal in appearance. X 8050.

11 July I967 EDIM INFECTION 49 9 AWA.f it- _... :ie.

12 so ADAMS AND KRAFT Vol. 5r, No. r FIG. IO. Somewhat higher-power view of infected epithelial cell showing more extensive involvement. Huge vesicles of endoplasmic reticulum comprise majority of cell volume and are filled with clumps of virus particles and irregular masses of lipid material. No alterations are seen in those cytoplasmic organelles that remain visible. Microvilli appear to be uninvolved. X I3,800.

13 July I 967 EDIM INFECTION A4-5I qwln- I hmo- i..s.,..,{ 10.1 r.;"iatv, Er 1.0 f,..14 li: s. tr, 1- L~,4_1.:.4.1i'a PO,'. 'AM16, t' -Wkt. d. ii:... -

14 52 ADAMS AND KRAFT Vol. 5.r, No. I FIG. II. Cross section of jejunal villus from infected mouse showing more severely involved cell at periphery of villus. This cell consists essentially of mass of vesicles containing large quantities of lipid material and clumps of virus particles. Few indentifiable organelles are seen. Mitochondria appear somewhat swollen, but there is minimal loss of cristae. Microvilli have been greatly attenuated. Both adjacent cells, though less severely involved, contain clumps of virus particles and dilated vesicles. X 8i6o.

15 July I967 EDIM INFECTION 53 11

16 54 ADAMS AND KRAFT Vol. 5I, No. z FIG. I2. Cross section of jejunal villus from infected mouse showing formation of microbullae and liberation of cytoplasmic contents, including ruptured vesicles, free virus particles, virus-containing vesicles, and virus-associated lipid material, into lumen of intestine. X I6,900.

17 July' I967 EDIM INFECTION : 77T i &. 12 or, :14r :.1 l.. A.. 11 Fi W,U O..W i..7t. V 1. 0 riab :.JR.4.z* V. il, 0

18 56 ADAMS AND KRAFT Vol. 5r, No. r Figures I3 to 22 show portions of cytoplasm of intestinal epithelial cells from infected mice showing 3 different particle types and varied membrane relationships of virus particles. FIG. I3. Portion of jejunal epithelium cell from infected mouse showing particularly well-developed dilated vesicle of rough-surfaced reticulum (lower center) containing virus-associated lipid and large number of virus particles. Some smaller virus-containing vesicles are present. No changes in nucleus, mitochondria, or microvilli are seen. X 24,000.

19 July 1967 EDIM INFECTION 57 13

20 ADAMS AND KRAFT Vol. 5.r, No. I FIG. I4. Virus particles of first type, which have a generally circular profile and a diameter of 8o m,u. Most are probably spherical. Each has outer membrane composed of typical double layer measuring about 70 A in width. Particles all located within vesicle of dilated rough-surfaced reticulum. Portion of cytoplasm (upper right) contains virus particle that impinges on membrane at surface of vesicle with protrusion into vesicle lumen (extreme right). X 84,ooo. FIG. I5. Dilated vesicle of rough-surfaced reticulum with single virus particle still attached to vesicle membrane and protruding almost entirely into vesicle lumen. Present within vesicle is irregular dumbbell-shaped mass of virus-associated lipid and numerous fine electron-dense strands of material. x 6o,ooo. FIG. 16. Two vesicles of granular reticulum. Vesicle on left contains virus particle whose outer membrane is still continuous with vesicle membrane. Vesicle on right contains lipid material and usual irregular electron-dense strands of material. Mitochondrion at top is normal in appearance. X 6o,ooo. FIG. I7. Vesicle of rough-surfaced reticulum containing "emerging" virus particle (top) and 3 virus particles of lesser diameter (second particle type in Fig. 20). X 56,ooo. FIG. I8. Virus particles within dilated vesicle of endoplasmic reticulum that is in continuity with outer membrane of nucleus in upper right. X 52,000. FIG. I9. Third type of particle, occurring only sporadically, consisted of elongated and ellipsoidal forms (center and right). Rarity of these forms suggests that they may not be functional biological forms but may represent aberrations of development. X 84,000. FIG. 20. Dilated vesicle of endoplasmic reticulum occupies lower two-thirds of figure. Rim of cytoplasm can be seen along top border. In addition to Type I particles (Fig. I5), there are numerous smaller particles that lack outer double membrane (Type 2 particles), measure 65 mu in diameter, and have slightly more irregular structure than Type I particle, suggesting that they may be abnormal forms. X 6o,ooo. FIG. 2I. Possible formation of virus particles within cytoplasm immediately adjacent to dilated vesicle of endoplasmic reticulum (left and extreme top center). Central portion of figure shows faint circular particles measuring about 50 mu in diameter interspersed in granular deposits of dense material. Granules in latter material are smaller than ribosomes, 3 or 4 clumps of which are at extreme right. Virus particles imping upon membrane of vesicle and protrude into its lumen. X 6o,ooo.

21 July I967 EDIM INFECTION * *-m W_ ^ l ,, 0. ~t ' *tv K b-...i,, qr i '... l~.-j. Ir Mvi.,..4 f IA..., ': i: : w h 20 21

22 4- X. -.. il p- 1. ".. ;..... s ;..7..L. *ie,. I I Y...t., ",.0-22 s i t., 4.,.R R.5 l's...:.' 5 {. i:,'.. $...s.. 4.S *'' tv K *23. D.> FIG. 22. Type 2 particles within vesicle of rough-surfaced reticulum. A thin rim of cytoplasm extends along right-hand border, and edge of mitochondrion is also visible (lower right). Several particles contain morphologic defects in their outer coat and protruding threads of material about 30 A in width, interpreted as viral nucleic acid. Some ribosomes stud surface of vesicle membrane traversing figure vertically along right side, thus identifying it as part of granular reticulum. X I68,ooo.