Pathogenesis of Rotavirus Infection in Mice

Transcription

1 INFECTION AND IMMUNITY, Nov. 1982, p Vol. 38, No /82/ $02.00/0 Copyright 1982, American Society for Microbiology Pathogenesis of Rotavirus Infection in Mice LYNN M. LITTLE't AND JOHN A. SHADDUCK2t* Departments of Microbiology1 and Pathology,2 University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas Received 2 February 1982/Accepted 29 July 1982 Three parameters of rotavirus infection, i.e., clinical disease, viral antigen in infected intestines, and infectious virus in feces, were assessed in infant mice nursed by mothers with or without preexisting rotavirus antibody. Diarrhea was the only consistent sign of clinical disease, and its course followed that of infection by about 1 day. Infected intestinal epithelial cells, except crypt cells, were observed by immunofluorescence microscopy in the duodenum, jejunum, ileum, and colon. Infection progressed in a proximal-to-distal direction with time. Viral antigen appeared in intestinal tissue later, was present in lower amounts, and disappeared sooner from infants nursed by mothers with preexisting rotavirus antibody, indicating that protection was passively transferred to these infants although the course of clinical disease was not changed. Rotavirus diarrhea in mice is a serious problem for mouse breeders and researchers because the virus is widespread, highly contagious, and highly resistant (18). Diarrhea during the first 2 weeks of life is the only consistent sign of disease. Affected mice normally make a full recovery. The disease was first described by Cheever and Mueller (6) in Kraft established the communicable and infectious nature of the agent in a series of investigations from 1957 through 1962 (15-18) and introduced the term epizootic diarrhea of infant mice. Adams and Kraft (2) confirmed by electron microscopy that the agent of epizootic diarrhea of infant mice is a virus and later (3) described the lesions of infected villous epithelial cells. Wilsnack et al. (41), using immunofluorescence, were able to detect rotavirus antigen in all regions of the intestine, but not in the stomach or liver. Rotavirus pathogenesis studies have been performed in calves (21, 22), pigs (7, 40), and lambs (35, 36), but these studies necessarily have been limited to outbred animals. The relationship of infection to clinical disease in rotavirus diarrhea still is poorly understood. The mouse (42; L. M. Little and J. A. Shadduck, Abstr. Int. Cong. Virol. 5th, Strassbourg, 1981, abstr. no. P16/14, p. 195) provides a convenient model for pathogenesis studies because the disease in mice has t Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, and Division of Infectious Diseases, The Children's Hospital Medical Center, Boston, MA t Present address: Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL high morbidity with low mortality, and large numbers of inbred animals can be used. In the current study, signs of clinical disease, viral antigen in infected tissue (detected by immunofluorescence assay of tissue sections), and infectious virus in feces (detected by immunofluorescence assay of infected tissue culture monolayers) were assessed in infants nursed by mothers with either preexisting detectable rotavirus antibody or no detectable serum antibody. MATERIALS AND METHODS Animals. Pregnant female BALB/c mice were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass. These mice and their offspring formed a breeding colony to provide animals for these experiments. Cells. MA-104 (fetal rhesus monkey kidney) cells, developed by Microbiological Associates, Bethesda, Md., were kindly provided to us by Mary K. Estes, Baylor College of Medicine, Houston, Tex. These were maintained in Eagle minimal essential medium supplemented with 5% fetal bovine serum and were seeded into chamber slides (Lab-Tek Products, Div. Miles Laboratories Inc., Naperville, Ill.) for virus titrations. Virus. Mouse rotavirus-infected emulsified infant mouse intestines were kindly supplied by John C. Parker, Microbiological Associates. Partially purified virus was prepared in our laboratory from infected infant intestines by a modification of the method of Much and Zajac (29). Partially purified virus from pooled feces of these mice was purified further as described below to insure that the virus preparation used for mouse inoculations did not contain another, possibly unknown, agent also capable of causing diarrheal disease in mice. Feces were layered on top of a 45% sucrose solution and centrifuged in an SW50.1 swinging bucket rotor at 755

2 756 LITTLE AND SHADDUCK 100,000 x g for 2.5 h in a Beckman L5-50 ultracentrifuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.) (4). The resulting pellet was suspended in 1 ml of 0.02 M Tris-hydrochloride buffer and extracted for 3 min in an equal volume of lipid solvent. This mixture was centrifuged at 1,300 x g for 10 min, and the aqueous phase was recovered, layered on top of a preformed, seven-step, 40-to-55% (wt/vol) CsCl gradient, and centrifuged in an SW41 swinging bucket rotor at 180,000 x g for 18 h in a Beckman L5-50 ultracentrifuge (32). Ten-drop fractions were collected, and buoyant densities were determined from refractive index readings. Electron microscopy of the fraction with a buoyant density of g/ml revealed rotavirus virions of typical appearance and no other virus-like particles. This material is termed highly purified virus. Highly purified virus was passaged once through infant mice to produce sufficient virus-rich inoculum for subsequent experiments. The resulting inoculum, a 20% (wt/vol) suspension of emulsified intestines termed passage 1 purified virus, contained 104-i times the 50% mouse infectious dose per ml. Virus-free control fluid for inoculation of control litters of mice was prepared as a 20% (wt/vol) suspension of emulsified intestines from infant mice fed virus-free CsCl of the same buoyant density (1.379 g/ml) as the highly purified virus. Production of antiserum. CsCl fractions, collected during gradient purification of virus, with buoyant densities (1.357 to g/ml) similar to those known for double capsid (1.36 g/ml) and single capsid (1.38 g/ml) rotavirus virions (24) were pooled and dialyzed against several changes of M Tris-hydrochloride buffer (32). A 1.2-ml volume of dialysate was injected into the marginal ear vein of a rabbit shown previously by immunofluorescence assay to be rotavirus antibody negative. After 14 days, an additional 1.2 ml of viruscontaining dialysate was mixed with an equal volume of Freund complete adjuvant and injected intramuscularly. The animal was exsanguinated 4.5 weeks later. The serum was adsorbed with mouse liver powder, filtered through a 0.22-p.m membrane filter to reduce background fluorescence, and tested by immunofluorescence assay on sections of an infected intestine. A sample was tested by a reference laboratory (Microbiological Associates) and found to be free of antibodies to 11 common viruses of mice. Infection of infant mice. A blood sample was collected from the ophthalmic venous plexus (31) of each dam, and rotavirus antibody titers were determined before the exposure of infant mice to virus. Litter size was limited to four infants per litter. All mice were given 5 p.l of either passage 1 purified virus or virus-free control fluid orally at 4 days of age. At this age, the mice were easier to handle than newborns, and preliminary experiments had shown that they still were fully susceptible to infection. Specimen collection. Two groups of litters were nursed by dams without preexisting rotavirus antibody (seronegative dams) and two by dams with preexisting rotavirus antibody (seropositive dams). For litters nursed by seronegative dams, feces and intestines of infants were collected from two animals per time point at 12-h intervals over the first 4 days after inoculation, daily over the next 4 days, and then on days 10, 12, 15, and 18. Control material was collected daily for the first 4 days and on days 6, 8, 12, and 18. A total of 32 INFECT. IMMUN. virus-infected and 8 control fluid-inoculated infant mice nursed by seronegative mothers were studied. For litters nursed by seropositive dams, the samples were collected from two animals per time point daily for the first 4 days and on days 6, 8, 12, and 18. Control material was collected on days 2, 4, 6, and 8. A total of 16 virus-infected and 4 control fluid-inoculated mice nursed by seropositive mothers were studied. At each collection, the infants within a litter were inspected for diarrhea after gentle palpation of their abdomens. Feces and the intestine of one infant were collected, as were feces from the dam. When the last littermate of a litter was removed, the dam was exsanguinated and her serum was obtained. Fecal specimens were diluted 1:10 in Hanks balanced salt solution and stored at -20 C before being processed. Sera were diluted 1:5 in phosphate-buffered saline and stored at -20 C. Each infant mouse was sacrificed by cervical dislocation and pinned, ventral side up, to a cork board. The abdomen was opened, and the entire intestine was lifted from the body cavity and stretched out to its full length on an ice-filled pan. By a modification of the procedure of Loria et al. (19), the intestine was coiled around the shaft of a 27-gauge needle protruding through the center of an aluminum foil-covered cardboard disk approximately 1 in. (2.54 cm) in diameter. Each intestine was immersed for 20 s in a beaker of 2- methylbutane submerged in a bath of liquid nitrogen for freezing at approximately -160 C and was cryostat sectioned in a plane to include all anatomical regions in each tissue section. Sections were transferred to glass slides which were fixed in acetone at room temperature for 10 min and stored at -70 C before examination by immunofluorescence microscopy. Immunofluorescence assay of intestinal sections. Three different primary sera were used on separate sections from each intestine: (i) anti-mouse rotavirus serum produced in a rabbit, (ii) anti-bovine rotavirus serum produced in a gnotobiotic pig, and (iii) antiporcine rotavirus serum also produced in a gnotobiotic pig. The latter two reference sera were the kind gift of E. H. Bohl, Ohio Agricultural Research and Development Center, Wooster, Ohio. Secondary sera were fluorescein-conjugated goat anti-rabbit immunoglobulin G (IgG) (Miles Laboratories, Inc., Elkhart, Ind.) and fluorescein-conjugated rabbit anti-porcine IgG (Miles). These were used at a high dilution to minimize background fluorescence. Added to each of the secondary sera was rhodamine-conjugated bovine serum albumin (Miles) to enhance the visibility of the tissue sections. Sections overlaid with primary sera were incubated at 37 C for 30 min in a moist chamber and then washed for 5 min in a phosphate-buffered saline bath by rotating on a rocker platform. After being air dried, the sections were overlaid with secondary sera and incubated again at 37 C for 30 min in a moist chamber. They were then washed for 5 min as before, allowed to air dry, and viewed under immersion oil with an Ortholux 2 epifluorescence microscope (Ernst Leitz Wetzlar GMBH, Germany). Specific fluorescence was scored in each anatomical region of the intestine (duodenum, jejunum, ileum, and colon) according to the following scheme: -, negative (no specific fluorescence seen); 1+, sparse fluorescence (less than 10% of the villi in one or more microscopic fields had fluorescing cells); 2+, scattered fluorescence (approximately 10 to 50% of the villi in

3 VOL. 38, 1982 ROTAVIRUS PATHOGENESIS 757 MoStwFe'{ FIG. 1. Rotavirus-infected sections of infant mouse intestines. Cryostat sections of intestines of infant mice infected with rotavirus were stained with immunofluorescence reagents as described in the text. The extent of infection is indicated by the percentage of villi with fluorescing cells in one or more microscopic fields. (A) 1 +, less than 10%; (B) 2+, approximately 10 to 50%; (C) 3+, approximately 50 to 90%; (D) 4+, approximately 90 to 100%. one or more microscopic fields had fluorescing cells); 3+, intermediate fluorescence (approximately 50 to 90% of the villi in one or more microscopic fields had fluorescing cells); and 4+, widespread fluorescence (approximately 90 to 100% of the villi in one or more microscopic fields had fluorescing cells). The amount of fluorescence was not estimated in a blind fashion; careful reference was made to known positive and negative control tissue sections with each set of slides studied. A typical microscopic field contained 50 to 100 villi. Examples of fields with 1+ to 4+ fluorescence are shown in Fig. 1.

4 758 LITTLE AND SHADDUCK /1 Fee ' Virus Shed is js42+ IInfant Dlorrhea ~4. ~,.Virus FIG.~ 2.Rtvrsifecso ~ nfn inifn0ienre Shed in Feces of Dams 10O Days After Inoculation FIG. 2. Rotavirus infection in infant mice nursed by seronegative dams. Infectious virus shed in feces of infants (O) and nursing dams (H) is given as the reciprocal of the average highest dilution of feces from two animals per time point capable of infecting MA- 104 cells as determined by immunofluorescence assay. Viral antigen in infant intestines (0) is given as the average amount of fluorescence (1 + to 4+) in all anatomical regions of intestines from two mice per time point. The vertical lines represent + 1 standard deviation of the mean. Infant diarrhea began by 48 h after inoculation, was present through day 8, and was absent by day 10. Virus assays. MA-104 were seeded into eight-well chamber slides at 100,000 cells per well in Eagle minimal essential medium containing gamma globulinfree fetal bovine serum (3%), penicillin, (200 U/ml), streptomycin (200,ug/ml), gentamicin (100,g/ml), and amphotericin B (0.1 plg/ml). Serial 1:10 dilutions of fecal specimens were made in chamber slide wells or microtiter plate wells beginning with a dilution of 102 because of the small quantities of feces from the youngest infants and to reduce toxicity. In some cases, dilutions were made immediately before cells were seeded into the wells. After the cells were seeded, the slides were incubated at 37'C for 4 h. All fluid was then removed by suction, the nascent cell sheets were washed with Hanks balanced salt solution, and fresh medium was added. For the more toxic specimens, the cells were seeded first and the slides were incubated for 4 h. Serial fecal dilutions then were made in the chamber slide wells, and the slides were returned to the incubator for 90 min. All fluid was then removed by suction, the cell sheets were washed with Hanks balanced salt solution, and fresh medium was placed in the wells. In all cases, chamber slides were removed from the incubator at 48 h after seeding. The medium was discarded, the chambers were removed, and the slides were fixed for 10 min in acetone at room temperature. The slides were held at -20'C before examination by immunofluorescence assay. This assay was performed as described above for intestinal sections except that the slides were washed for 15 min instead of 5 min. The virus titer of each specimen was the highest fecal dilution yielding fluorescing cells. Titration of sera. Sections of mouse intestines infected with passage 1 purified virus were used. Sera were diluted 1:5, 1:10, 1:20, and in increments of 1:20. INFECT. IMMUN. Titers were determined by indirect immunofluorescence assay with dilutions of sera from dams as the primary sera and fluorescein-conjugated goat antimouse IgG (Cappel Laboratories, Cochranville, Penn.) plus rhodamine-conjugated bovine serum albumin as the secondary serum. The assay was performed as described above for intestinal sections of infected infants. Serum titer endpoints were the highest dilutions at which specific fluorescence of epithelial cells still was clearly visible. RESULTS Diarrhea in infant mice. The course of diarrhea was the same in litters nursed by either seronegative or seropositive dams. Diarrhea began by 48 h post-inoculation (p.i.), was present in both groups through 8 days p.i. and absent by 10 days p.i. Diarrhea never was seen in nursing dams. The severity of the disease seemed to vary with the litter. Features of disease other than diarrhea included lethargy and distended abdomens. Also, the quantity of feces observed varied both from litter to litter and from time to time within the same litter. Because of this variability, the severity of the disease could not be quantitated for comparison between litters nursed by seronegative and seropositive dams. Signs of illness did not occur in infants given virus-free control fluid. Viral antigen in intestinal tissue. Rotaviruses of various species cross-react serologically (43). The hyperimmune reference sera to bovine and porcine rotavirus antigens provided a ready means for assessing the quality of the rabbit immune serum prepared against mouse rotavirus antigens. The patterns of fluorescence produced by the three sera were the same at all times studied. Specific fluorescence due to viral antigen was associated only with the cytoplasm of epithelial cells lining the intestine. Crypt cells never were seen to fluoresce. The number of fluorescing cells increased with proximity to the villous tip, but it is not known whether infectivity was due to cell differentiation or simply to duration on the villus. Fluorescence was first seen at 24 h p.i. in both animals nursed by seronegative dams but in only one of two animals nursed by seropositive dams. The amount of specific fluorescence was greater in proximal than in distal regions at this time. Fluorescence was widespread in both groups by 48 h p.i., and it continued to be intermediate to widespread in animals nursed by initially seronegative dams through 6 to 7 days p.i. (Fig. 2). In contrast, after 48 h the amount of specific fluorescence was diminished considerably in animals nursed by seropositive dams (Fig. 3). By 72 h p.i., the amount of fluorescence in intestines of mice nursed by these mothers already was less than that in mice nursed by initially

5 VOL. 38, 1982 ti 'B 1O L * \ Viral Antigen in Infn Intesi 103 I-.nfat Ciorrs r / 1+ A Days After Inoculation FIG. 3. Rotavirus infection in infant mice nursed by seropositive dams. Infectious virus shed in feces of infants (I) is given as the reciprocal of the average highest dilution of feces from two animals- per time point capable of infecting MA-104 cells as determined by immunofluorescence assay. Virus was not detected in feces of seropositive nursing dams. Viral antigen in infant intestines (0) is given as the average amount of fluorescence (1+ to 4+) in all anatomial regions of intestines from two mice per time point. The vertical lines represent + 1 standard deviation of the mean. (The absence of fluorescence in the intestine of one mouse at 24 h p.i. accounts for the high standard deviation at this time.) Infant diarrhea began by 48 h after inoculation, was present through day 8, and was absent by day 10. seronegative dams at 7 days p.i. Fluorescence was gone in the former group at 8 days p.i., but remained in the ileum and colon of the latter group. As it had begun, the infection subsided in a proximal-to-distal direction (Fig. 4). No fluorescence was seen in intestines from either group after 8 days p.i. Fluorescence never was observed in intestines of infants given virus-free control fluid. Infectious virus in feces. Infants nursed by either seronegative (Fig. 2) or seropositive (Fig. 3) dams produced feces that were infectious at a 10-4 dilution at 24 h p.i. At 48 h p.i., feces from both groups were infectious at dilutions as high as 10-5 or By 72 h and thereafter through 6 days p.i., feces from infants of seronegative dams were an average of 0.5 logl0 to 1 logl0 more infectious. Infectious virus no longer was detectable in feces of infants of seropositive dams at 8 days p.i., whereas feces of infants of initially seronegative dams still contained infectious virus at dilutions as high as 10-4 or The only maternal feces that contained detectable virus were those of seronegative dams, and these were positive only from 60 through 96 h postexposure. Infectious virus never was found in feces of infants or dams inoculated with virusfree control fluid. H ROTAVIRUS PATHOGENESIS 759 Antibody titers of sera from dams. Serum antibody titers of initially seronegative mothers had not begun to rise by 96 h postexposure (Table 1). By this time their infants had had diarrhea for 2 days and had had both infected intestinal epithelial cells and infectious virus in feces for 3 days. By day 8, when infection was waning in the infants, the antibody titers of these dams had risen substantially. In contrast, in dams with preexisting rotavirus antibody titers, a rise in titer already was evident at 96 h postexposure. At day 18, the antibody titers in this group were more than twice those in initially seronegative dams. The titers of dams nursing infants fed virus-free control fluid did not change. DISCUSSION Only the epithelial cells lining the intestines of infant mice, not the immature, undifferentiated epithelial cells residing in crypts between the villi, were susceptible to rotavirus infection. Using electron microscopy, Adams and Kraft (3) have described the lesions of infected cells and predicted a gradient of susceptibility increasing from crypt to villous tip, but they did not report whether they have observed infected cells actually within the crypts. Similarly, Wilsnack et al. (41) have noted that viral antigen is limited to epithelial cells and lumen of the intestinal tract, but they did not state whether infected cells are restricted to villi or also are present in crypts. Identification of the target cell of an infection is important for an understanding of the pathogenesis of the disease. In both transmissible gastroenteritis of swine and feline panleukopenia, diarrhea results from the depletion of villous epithelial cells. The pathogenesis of the lesion in the two diseases differs, however. In transmissible gastroenteritis, villous cells are destroyed by the virus, but because crypt epithelial cells are not damaged, the lesion is rapidly and completely reversible (12, 28). In contrast, in feline panleukopenia, the crypt cells themselves are destroyed by the virus. Migration and shedding of villous cells continues, and because these cells cannot be replaced by the damaged crypt cells, the villi become denuded, the lesion remains, and the absorptive capacity of the intestine is lost (28). The finding in the present study that crypt cells are spared in rotavirus infection suggests a pathogenic mechanism similar to that of transmissible gastroenteritis rather than feline panleukopenia (27). Although the time course of clinical disease in mice has been established (6, 15), the time of first appearance of viral antigen in all anatomical regions of the intestine (duodenum, jejunum, ileum, and colon) and its distribution throughout the course of infection have not been reported

6 760 LITTLE AND SHADDUCK INFECT. IMMUN. S I r B 4--) 12h 24h 3Gh 48h G0h 72h 84h 9Gh 5d Gd 7d Xd 10d Time After Inoculation FIG. 4. Location of rotavirus antigen in intestines of infant mice nursed by (A) seronegative or (B) seropositive dams. Viral antigen is expressed as the average amount of fluorescence (1+ to 4+) in each anatomical region of the intestine, i.e., duodenum (0), jejunum (0), ileum (O), and colon (U), from two mice per time point (except panel B, 24 h, for which data are from one mouse only). The vertical lines represent + 1 standard deviation of the mean. previously. Antigen first appeared at 24 h after inoculation and persisted in intestinal tissue through days 6 to 8. It is interesting to note that the course of diarrhea followed the course of viral antigen in tissue and of infectious virus in feces by about 1 day. Thus, the disease did not begin immediately, but required about 24 h to develop after the onset of infection. Similarly, once infection no longer was detectable at the tissue level, repair of the absorptive cell layer sufficient to restore normal transport of fluids across the lumenal surface and end the diarrhea apparently required a like amount of time. All anatomical regions of the infant mouse intestine were susceptible to rotavirus infection. This is in contrast to the findings of others that rotavirus infection is confined to epithelial cells of the small intestine in humans (25), calves (21), and pigs (20). Small numbers of infected cells also have been found in the epithelium of the colon and cecum of lambs (35) and, in one study, in the colon of pigs (40). The absence of evidence of infection does not necessarily mean that epithelial cells of the lower bowel are resistant. Physical factors may help to account for the concentration of infection in the upper regions of the intestinal tract in these species. For example, the number of susceptible cells available per infectious virion may be much higher in the small intestine of larger animals because of the much greater size of the intestine. The infection may have run its course before the supply of locally available cells has been depleted. Also, the rate at which intestinal contents are transported may differ. Kraft (16) has found that India ink fed to infant mice already is present in the

7 VOL. 38, 1982 TABLE 1. ROTAVIRUS PATHOGENESIS 761 Serum antibody titers' to mouse rotavirus antigen in dams of infected litters Titer in seronegative dams of the following Titer in seropositive dams of the following Time of bleeding litters: litters: (day p.i.) Uninfected Uninfected Group 1 Group 2 control Group 1 Group 2 control Pre <1:5 <1:5 2 <1:5 <1:5 Pre <1:5 <1:5 <1:5 1:20 1:100 4 <1:5 <1:5 <1:5 1:40 1:120 Pre <1:5 <1:5 1:20 8 1:140 1:60 1:20 Pre <1:5 <1:5 <1:5 1:60 1: :100 1:80 <1:5 1:260 1:180 atiters were determined by indirect immunofluorescence assay as described in the text. Each pair of ratios represents the titers of two sera from a single dam, a preserum collected before feeding of rotavirus to infants and a second (exsanguination) serum collected at the time shown. Ten seronegative and five seropositive dams were included in the study. rectum 3 h later. In larger animals, intestinal contents may be transported more slowly. The response of epithelial cells to rotavirus introduced directly into the large bowel has not been investigated. Viral antigen did not appear in all anatomical regions simultaneously. Rather, there was a definite proximal-to-distal progression of infection with time (Fig. 4). Similar results have been found by others in infections with calf rotavirus (22, 38) and pig rotavirus (10) and with human rotavirus infections of calves (23) and monkeys (45). A similar progression of infection has not been found, however, by others studying rotavirus infections of lambs (35) and pigs (30), perhaps because progression of the infection is more difficult to detect when the entire intestinal tract does not become infected. It should be appreciated that in the current study not all cells within any anatomical region fluoresced. Rather, fluorescing cells normally occupied less than 10%, up to perhaps 50%, of the total area of any anatomical region in the intestinal sections viewed. The number of fluorescing cells per villus corresponded roughly with the number of infected villi (1 + to 4+) per microscopic field. The reason for the discontinuous nature of the infection is not known, but it is possible that the infection could be initiated only after the chance encounter of infectious virions with cells at a time when they were in a phase of the cell cycle permissive for viral expression (26). Cells so infected could be expected to produce large numbers of new virions which, once released, would then be amplified for infection of other permissive cells in the same general area. The infection would then be likely to progress from the local site in a distal direction, moving along with the flow of intestinal contents. Infected cells usually were observed halfway up the villus and above. An abundance of infected cells nearer the villous base occurs in other species, such as the calf, in which the disease is more severe (27). It is possible that in each species infection is concentrated in those villous cells which have the greatest amount of brush border enzymes. Holmes et al. (11) have proposed that lactase acts as a combined receptor and uncoating enzyme for rotavirus. This, however, would restrict infection to those cells with lactase on their surface. In conflict with this is our finding in mice of extensive infection of colonic epithelial cells, which contain almost no enzymes (9). The distance of infected cells in the colon from the pancreas also challenges the proposal by Theil et al. (39) that pancreatic enzymes facilitate viral attachment and penetration. It is possible, of course, that these proposed mechanisms are functional but not required for infection. Schoub et al. (34), using electron microscopy, could detect virus for at least 3 weeks in feces of dams exposed to rotavirus-infected litters. The number of virus particles diminished greatly once diarrhea was resolved in their infants. The proportion of shed particles that were infectious was not determined. The results of the current study indicated that high levels of infectious virus were not shed by the infants beyond the time when they were clinically ill. Thus, if a carrier state exists in either recovered infants or their mothers, the quantities of infectious virus shed probably are low. Recently, Wolf et al. (42), studying the effects of corticosteroids on susceptibility to rotavirus

8 762 LITTLE AND SHADDUCK infection, have reported that the presence or absence of antibody in mothers did not influence the susceptibility of their infants. In contrast, in the current study, antigen appeared later, was present in tissue in lower amounts, and disappeared sooner from infants nursed by mothers with preexisting rotavirus antibody. The quantities of infectious virus shed in feces were lower, and detectable virus disappeared sooner from infants nursed by dams with preexisting antibody. These findings indicate that protection at the tissue level is afforded infants suckled by seropositive dams, even though that protection is not adequate to alter the clinical course of disease. Observations on rotavirus infection of calves (44), lambs (37), and pigs (33) and on transmissible gastroenteritis of swine (1, 5) have revealed that intestinal immunity is not related to serum antibodies but rather to the presence of secretory antibodies in the intestinal lumen. Similarly, in humans, the presence of preexisting serum antibody to rotavirus does not correlate with resistance to infection in children or adults (13, 14). Secretory antibodies may be protective whether they are produced locally or ingested in colostrum or milk. In sows (5) and ewes (37), antibodies appear in milk after oral or intramammary, but not after intramuscular, immunization. Using an immunofluorescence assay, Schoub et al. (34) could not find antibodies to rotavirus antigen in colostrum or milk of mother mice nursing rotavirus-infected litters. These authors also found little or no rotavirus antibody in human colostrum and milk despite adequate amounts of IgA and serum antibody. Using the more sensitive methods of enzymelinked immunosorbent assay (46) and radioimmunoassay (8), however, others have shown that antirotavirus secretory IgA is present in colostrum and persists in milk of human mothers for many months. Since mother mice ingest a continual dose of rotavirus antigen while constantly cleaning their diarrheal young, it seems likely that antirotavirus antibody is present in mouse milk during the course of infection in the infants. The fact that the clinical course of disease was not diminished in mice nursed by seropositive dams may indicate that although protection was conferred at the tissue level, it was not adequate to spare enough absorptive cells from infection for the maintenance of normal fluid balance between tissue and lumen. A time course analysis of rotavirus antigen in the intestinal tissue of other species may reveal similar protection at the tissue level. A more thorough understanding of the factors involved in mucosal immunity and of the pathogenesis of rotavirus infections in INFECT. IMMUN. various species may lead to more effective means of immunization so that cellular protection can be translated into clinical protection. ACKNOWLEDGMENTS The excellent technical assistance of Ann Fields, Mary Jean Geroulo, George Lawton, Elizabeth Mayhew, and Nancy Sisco is acknowledged. This work was supported by Public Health Service grant RR00890 from the National Institutes of Health. LITERATURE CITED 1. Abou-Youssef, M. H., and M. Ristic Protective effect of immunoglobulins in serum and milk of sows exposed to transmissible gastroenteritis virus. Can. J. Comp. Med. 39: Adams, W. R., and L. M. Kraft Epizootic diarrhea of infant mice: identification of the etiologic agent. Science 141: Adams, W. R., and L. M. Kraft Electron-microscopic study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus). Am. J. Pathol. 51: Bishop, R. F., G. P. Davidson, I. H. Holmes, and B. J. Ruck Detection of a new virus by electron microscopy of faecal extracts from children with acute gastroenteritis. Lancet i: Bohl, E. H., and L. J. Saif Passive immunity in transmissible gastroenteritis of swine: immunoglobulin characteristics of antibodies in milk after inoculating virus by different routes. Infect. Immun. 11: Cheever, F. S., and J. H. Mueller Epidemic diarrheal disease of suckling mice. III. The effect of strain, litter, and season upon the incidence of the disease. J. Exp. Med. 88: Crouch, C. R., and G. N. Woode Serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus. J. Med. Microbiol. 11: Cukor, G., N. R. Blacklow, F. E. Capozza, Z. F. K. Panjvani, and F. Bednarek Persistence of antibodies to rotavirus in human milk. J. Clin. Microbiol. 9: Guyton, A. C Basic human physiology: normal function and mechanisms of disease, p W. B. Saunders Co., Philadelphia. 10. Hall, G. A., J. C. Bridger, R. L. Chandler, and G. N. Woode Gnotobiotic piglets experimentally infected with neonatal calf diarrhea reovirus-like agent (rotavirus). Vet. Pathol. 13: Holmes, I. H., S. M. Rodger, R. D. Schnagl, and B. J. Ruck Is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? Lancet i: Hooper, E. E., and E. 0. Haelterman Lesions of the gastrointestinal tract of pigs infected with transmissible gastroenteritis. Can. J. Comp. Med. 33: Kapikian, A. Z., H. W. Kim, R. G. Wyatt, W. L. Cline, J. 0. Arrobio, C. D. Brandt, W. J. Rodriguez, D. A. Sack, R. M. Chanock, and R. H. Parrott Human reovirus-like agent as the major pathogen associated with "winter" gastroenteritis in hospitalized infants and young children. N. Engl. J. Med. 294: Kim, H. W., C. D. Brandt, A. Z. Kapikian, R. G. Wyatt, J. 0. Arrobio, W. J. Rodrigiez, R. M. Chanock, and R. H. Parrott Human reovirus-like agent infection. Occurrence in adult contacts of pediatric patients with gastroenteritis. J. Am. Med. Assoc. 238: Kraft, L. M Studies on the etiology and transmission of epidemic diarrhea of infant mice. J. Exp. Med. 101: Kraft, L. M Observations on the control and natural history of epidemic diarrhea of infant mice (EDIM). Yale J. Biol. Med. 31: Kraft, L. M Responses of the mouse to the virus of

9 VOL. 38, 1982 epidemic diarrhea of infant mice. Neutralizing antibodies and carrier state. Proc. Anim. Care Panel 11: Kraft, L. M Two viruses causing diarrhoea in infant mice, p In R. J. C. Harris (ed.), The problems of laboratory animal disease. Academic Press, Inc., New York. 19. Loria, R. M., S. Kibrick, A. El-Bermani, and S. Broitman Preparation of intestine and other elongated specimens for histologic and immunofluorescent studies. Am. J. Clin. Pathol. 60: McAdaragh, J. P., M. E. Bergeland, R. C. Meyer, M. W. Johnshoy, I. J. Stotz, D. A. Benfield, and R. Hammer Pathogenesis of rotaviral enteritis in gnotobiotic pigs: a microscopic study. Am. J. Vet. Res. 41: Mebus, C. A., and L. E. Newman Scanning electron, light and immunofluorescent microscopy of intestine of gnotobiotic calf infected with reovirus-like agent. Am. J. Vet. Res. 38: Mebus, C. A., E. L. Stair, N. R. Underdahl, and M. J. Twiehaus Pathology of neonatal calf diarrhea induced by a reo-like virus. Vet. Pathol. 8: Mebus, C. A., R. G. Wyatt, R. L. Sharpee, M. M. Sereno, A. R. Kalica, A. Z. Kapikian, and M. J. Twiehaus Diarrhea in gnotobiotic calves caused by the reovirus-like agent of human infantile gastroenteritis. Infect. Immun. 14: Middleton, P. J Rotavirus: clinical observations and diagnosis of gastroenteritis, p In E. Kurstak and C. Kurstak, (ed.), Comparative diagnosis of viral diseases, vol. 1A. Academic Press, Inc., New York. 25. Middleton, P. J., M. T. Szymanski, G. D. Abbott, R. Bortolussi, and J. R. Hamilton Orbivirus acute gastroenteritis of infancy. Lancet i: Misra, V., and L. A. Babiuk Possible mechanism of rotavirus persistence. J. Supramol. Struct. 12(Suppl. 4): Moon, H. W Mechanisms in the pathogenesis of diarrhea: a review. J. Am. Vet. Med. Assoc. 172: Moon, H. W., J. 0. Norman, and G. Lambert Agedependent resistance to transmissible gastroenteritis of swine (TGE). I. Clinical signs and some mucosal dimensions in small intestine. Can. J. Comp. Med. 37: Much, D. H., and I. Zajac Purification and characterization of epizootic diarrhea of infant mice virus. Infect. Immun. 6: Pearson, G. R., and M. S. McNulty Pathological changes in the small intestine of neonatal pigs infected with a pig reovirus-like agent (rotavirus). J. Comp. Pathol. 87: Riley, V Adaptation of orbital bleeding technic to rapid serial blood studies. Proc. Soc. Exp. Biol. Med. 104: Rodger, S. M., R. D. Schnagl, and I. H. Holmes Biochemical and biophysical characteristics of diarrhea viruses of human and calf origin. J. Virol. 16: ROTAVIRUS PATHOGENESIS Saif, L. J., and E. H. Bohl Role of secretory IgA in passive immunity of swine to enteric viral infections, p In P. L. Ogra and D. Dayton (ed.), Immunology of breast milk. Raven Press, New York. 34. Schoub, B. D., 0. W. Prozesky, G. Lecatsas, and R. Oosthuizen The role of breast feeding in the prevention of rotavirus infection. J. Med. Microbiol. 11: Snodgrass, D. R., K. W. Angus, and E. W. Gray Rotavirus infection in lambs: pathogenesis and pathology. Arch. Virol. 55: Snodgrass, D. R., A. Ferguson, R. Allan, K. W. Angus, and B. Mitchell Small intestinal morphology and epithelial cell kinetics in lamb rotavirus infections. Gastroenterology 76: Snodgrass, D. R., and P. W. Wells Rotavirus infection in lambs: studies on passive protection. Arch. Virol. 52: Stair, E. L., C. A. Mebus, M. J. Twiehaus, and N. R. Underdahl Electron microscopy of intestines infected with a reovirus-like agent. Vet. Pathol. 10: Theil, K. W., E. H. Bohl, and A. G. Agnes Cell culture propagation of porcine rotavirus (reovirus-like agent). Am. J. Vet. Res. 38: Theil, K. W., E. H. Bohl, R. F. Cross, E. M. Kohler, and A. G. Agnes Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs. Am. J. Vet. Res. 39: Wilsnack, R. E., J. H. Blackwell, and J. C. Parker Identification of an agent of epizootic diarrhea of infant mice by immunofluorescent and complement-fixation tests. Am. J. Vet. Res. 30: Wolf, J. L., G. Cukor, N. R. Blacklow, R. Dambrauskas, and J. S. Trier Susceptibility of mice to rotavirus infection: effects of age and administration of corticosteroids. Infect. Immun. 33: Woode, G. N., J. C. Bridger, J. M. Jones, T. H. Flewett, A. S. Bryden, H. A. Davies, and G. B. B. White Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals. Infect. Immun. 14: Woode, G. N., J. Jones, and J. C. Bridger Levels of colostral antibodies against neonatal calf diarrhea virus. Vet. Rec. 97: Wyatt, R. G., D. L. Sly, W. T. London, A. E. Palmer, A. R. Kalica, D. H. Vankirk, R. M. Chanock, and A. Z. Kapikian Induction of diarrhea in colostrum-deprived newborn rhesus monkeys with the human reoviruslike agent of infantile gastroenteritis. Arch. Virol. 50: Yolken, R. H., R. G. Wyatt, L. Mata, J. J. Urrutia, B. Garcia, R. M. Chanock, and A. Z. Kapikian Secretory antibody directed against rotavirus in human milkmeasurement by means of enzyme-linked immunosorbent assay. J. Pediatr. 93: