INTRODUCTION

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Rotavirus causes acute diarrheal disease by infecting villous epithelial cells of the small intestine. Unfortunately, in vitro studies of interactions between rotavirus and intestinal epithelial cells (IEC) have been limited by the lack of established small intestinal cell lines. Studies of rotavirus using MA104 cells (monkey kidney epithelial cells), Madin-Darby canine kidney cells (MDCK cells) (13), and human colonic carcinoma cell lines, particularly Caco-2 and HT-29 cells (12, 13, 16, 17, 30), have been carried out to investigate rotavirus-epithelial cell interactions in vitro. However, the utility of these cell lines is limited by the fact that they (i) are not derived from the small intestine, (ii) lack major histocompatibility complex (MHC) compatibility with a variety of genetically defined strains of experimental animals, and (iii) are malignantly transformed (colonic carcinoma cell lines). Although immortalized murine small intestinal cell lines have been established (e.g., m-IC_cl2), they have not previously been studied for susceptibility to rotavirus (4, 31, 33).

We developed a method to cultivate primary murine small IEC in vitro. We found that primary IEC proliferated in culture, remained viable for 3 weeks, and maintained expression of cytokeratin, alkaline phosphatase, and class II antigens, characteristic of IEC. Primary cultured murine small IEC supported the growth of rotavirus to a greater extent than the murine small intestinal cell line, m-IC_cl2.

	MATERIALS AND METHODS

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Animals. Male and female 5- to 8-week-old BALB/c mice were obtained from Taconic Breeding Laboratories (Germantown, N.Y.) and were mated. At 7 days, BALB/c mice were sacrificed by cervical dislocation, and their small intestines were removed.

Epithelial cell isolation and culture. IEC were isolated by using a modification of the method of Evans and coworkers (10). Small intestines were opened longitudinally and were washed in Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution (HBSS) (Gibco, Gaithersburg, Md.) containing 2% glucose, 25 ng of amphotericin B per ml, 100 U of penicillin per ml, and 100 µg of streptomycin per ml. By using a scapel blade, intestines were cut into 1-mm fragments and were incubated for 10 min at 22°C on a shaker platform in Ca²⁺- and Mg²⁺-free HBSS containing 60 U of collagenase XIa (Sigma, St. Louis, Mo.) per ml, 0.02 mg of dispase I (Boehringer Mannheim, Indianapolis, Ind.) per ml, 2% bovine serum albumin, and 0.2 mg of soybean trypsin inhibitor (Sigma) per ml. Cells and small sheets of intestinal epithelium were separated from the denser intestinal fragments by harvesting supernatants after two 60-s sedimentations at 1 × g in medium containing Dulbecco's modified Eagle medium (DMEM) (Gibco), 10% sorbitol (Gibco), 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 5% fetal bovine serum (FBS) (Biowhittaker, Walkerville, Md.). Cells were centrifuged five times at 120 × g for 3 min in DMEM plus 2% sorbitol. Supernatant fluids containing monodispersed IEC, nonepithelial cells, and debris were discarded. The remaining pellet consisted predominantly of cells in intact crypts and small sheets of intestinal epithelium (organoids). Cell viability was assessed by trypan blue exclusion and light microscopy. Morphology was determined by phase-contrast microscopy after staining with hematoxylin and eosin. Cells were cultured in 24-well plates, using cells from five mice per plate, at a seeding density of approximately 200 to 300 organoids/cm². One hour before plating cells, culture surfaces were coated with 40 µl of Matrigel (Collaborative Biomedical Products, Bedford, Mass.) per cm² diluted 1:2 in phenol-red-free DMEM (Sigma). For immunohistochemical studies, cells were grown on glass coverslips coated with Vectabond (Vector Laboratories, Burlingame, Calif.) prior to coating with Matrigel. Epithelial cells were cultured in epithelial cell medium (ECM) containing equal volumes of phenol-red-free DMEM and Ham's F-12 medium (Biowhittaker) with the following additives: 5 µg of insulin (Sigma) per ml, 5 × 10⁸ M dexamethasone (Sigma), 60 nM selenium (Sigma), 5 µg of transferrin (Sigma) per ml, 5 × 10⁸ M triiodothyronine (Sigma), 10 ng of epidermal growth factor (Sigma) per ml, 20 mM HEPES, 2 mM glutamine, 100 U of penicillin per ml, 100 µg of streptomycin per ml, 0.2% D-glucose, and 2% FBS. ECM was used within 2 weeks of preparation to ensure that the activity of growth factors was maintained. Cells were cultured in 5% CO₂ at 37°C with periodic supplementation of medium to maintain a volume of 2 ml per well. Cell growth and morphology were assessed by phase microscopy.

Cell lines. m-IC_cl2 cells, a simian-virus-40-transformed murine small intestinal cell line that maintains a crypt phenotype (4), were kindly provided by Alain Vandewalle (Institut National de la Santé et de la Recherche Médicale, Paris, France) and were grown in 25-cm²-volume culture flasks in ECM. For enzyme analysis, cells were grown in flasks coated with 5 µg of rat tail collagen per cm² (Collaborative Biomedical Products). m-IC_cl2 cells passaged 50 to 60 times were studied. 3T3 cells (murine fibroblast cells; American Type Culture Collection), human intestinal smooth muscle cells (American Type Culture Collection), and MA104 cells (monkey kidney epithelial cells) were grown in DMEM containing 10% FBS, 100 U of penicillin per ml, and 100 µg of streptomycin per ml (DMEM-C).

Virus. Rhesus rotavirus (RRV) (Nathalie Schmidt, Berkeley, Calif.) was grown in MA104 cells and quantitated as previously described (20). Wild-type murine rotavirus EDIM (Richard Ward, Children's Hospital Research Foundation, Cincinnati, Ohio) was inoculated orally into suckling Swiss Webster mice and was prepared as an intestinal homogenate as previously described (20). Murine rotavirus EDIM adapted to growth in MA104 cells after nine passages in vitro was also obtained from Richard Ward.

Immunohistochemical studies of small IEC. Freshly isolated epithelial cells were attached to glass slides by using a cytocentrifuge (Shandon, Inc., Pittsburgh, Pa.). Primary small intestinal cells grown on coverslips were gently washed twice with serum-free ECM and were fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 60 min. Coverslips were mounted on slides and stored at 80°C. Alternatively, cells were directly stained in 24-well plates after fixation. All cells were permeabilized with 0.04% Triton-X 100 for 15 min and were blocked with 2% rat serum in PBS. Cells were incubated with either (i) rabbit polyclonal wide-spectrum anticytokeratin (DAKO, Carpentaria, Calif.) diluted 1:100 in PBS with 2% rat serum, (ii) monoclonal mouse anti--smooth-muscle actin (Sigma) diluted 1:200, or (iii) monoclonal mouse antivimentin (Sigma) diluted 1:100. Secondary antibodies were either mouse anti-rabbit immunoglobulin G (IgG), goat anti-mouse IgG, or goat anti-mouse IgM (Sigma) conjugated to fluorescein isothiocyanate (FITC) and diluted 1:100 in PBS with 2% rat serum.

Assessment of primary epithelial cell viability and growth. Primary epithelial cell cultures were analyzed for viability by enzymatic conversion of calcein AM to green fluorescent calcein and exclusion of ethidium homodimer (red fluorescence) from the cell nucleus (Live/Dead Viability/Cytotoxicity Kit; Molecular Probes, Eugene, Oreg.). The number of dead cells per high-power field was counted at various intervals after plating. Synthesis of DNA was assessed by uptake of [³H]thymidine (Amersham, Arlington Heights, Ill.). Briefly, 2 µCi of [³H]thymidine was added to approximately 5 × 10⁴ cultured IEC 2, 4, 7, 10, or 14 days after plating. After incubation for 5 h in 5% CO₂ at 37°C, cultures were frozen at 20°C. Cell contents were harvested by freezing and thawing samples three times, and samples were analyzed for incorporation of [³H]thymidine by using a -scintillation counter.

Determination of disaccharidase expression by IEC. Expression of the disaccharidases sucrase, maltase, lactase, and palatinase was determined by using a modification of the substrate-reduction method of Dahlqvist (9). The following cells were assayed: freshly isolated IEC from suckling mice, obtained prior to plating; cultured primary IEC, obtained 6 to 19 days after plating and detached from the culture surface by incubation in 0.5% (wt/vol) trypsin-EDTA for 5 min at 37°C; m-IC_cl2 cells cultured on rat tail collagen; and 3T3 fibroblasts. All cells were washed twice in PBS, were pelleted, and were stored at 20°C. Expression of alkaline phosphatase in freshly isolated IEC, primary IEC cultures, and cell lines was examined by using the Vector Red Alkaline Phosphatase substrate kit (Vector Laboratories). The substrate reagent, prepared with the addition of 125 mM levamisole (Vector Laboratories), was specific for the intestinal isoenzyme of alkaline phosphatase. Cells were incubated in substrate reagent for 20 min at room temperature, were washed with 100 mM Tris-HCl (pH 8.2), and were examined for expression of alkaline phosphatase by fluorescent microscopy.

Flow cytometric analysis of IEC for MHC class II antigen expression. To determine the expression of MHC class II antigens by IEC, cells were digested from intestinal fragments of 7- to 10-day-old-mice as described above and were analyzed by flow cytometry. To confirm the epithelial nature of the cells, expression of cytokeratin was also determined by using two-color flow cytometry. Digestion of adult IEC was performed by incubation of cells for 30 min at 22°C in Ca²⁺- and Mg²⁺-free HBSS containing 300 U of collagenase XIa (Sigma) per ml, 0.1 mg of dispase I (Boehringer Mannheim) per ml, 2% bovine serum albumin, and 0.2 mg of soybean trypsin inhibitor per ml. Digested cells were centrifuged once in DMEM with 2% sorbitol at 120 × g for 3 min, and a single-cell suspension was obtained from supernatant fluids. Splenic B cells obtained from suckling and adult mice and m-IC_cl2 cells were analyzed as controls. All cells were treated for 30 min at 4°C with DMEM-C containing 5 µg of mouse immunoglobulin (Jackson Laboratories, West Grove, Pa.) per ml and were incubated for 45 min at 4°C with either 5 µg of mouse anti-mouse Ia^d conjugated to biotin (Pharmingen, San Diego, Calif.) per ml or 5 µg of mouse anti-mouse Ia^k conjugated to biotin (Pharmingen) per ml as the control antibody, in DMEM-C. After washing, cells were incubated for 30 min in 2.5 µg of streptavidin conjugated to phycoerythrin (Pharmingen) per ml. Following surface staining, cells were fixed and permeabilized for 30 min at 4°C in 0.1% paraformaldehyde in DMEM-C, were washed, and were incubated for 60 min at 4°C with either rabbit anti-cow broad-spectrum cytokeratin (DAKO), or nonimmune rabbit serum (provided by H. F. Clark, Philadelphia, Pa.), diluted 1:50 in DMEM-C. Mouse anti-rabbit IgG conjugated to FITC (Sigma) and diluted 1:100 in DMEM-C was then applied for 30 min.

Rotavirus infection of cultured murine small IEC and m-IC_cl2 cells. Primary IEC cultures from suckling mice (obtained 7 to 14 days after plating) and confluent m-IC_cl2 cells, grown in 24-well plates, were washed twice in serum-free ECM and were overlaid for 60 min at 37°C with 100 µl of either RRV, EDIM intestinal homogenate (diluted 1:10 in serum-free ECM and filtered through a 0.45-µm-pore-size filter), or cell-culture-adapted EDIM strain per well. Trypsin (0.2 µg/ml) was added to each viral overlay. Infections were performed by using a multiplicity of infection (MOI) of 1 for immunofluorescence studies and an MOI of 10 for determinations of viral growth using RRV. Control cultures were overlaid with mock-infected suspensions of MA104 cells or intestinal homogenate obtained from non-rotavirus-infected mice. After incubation with virus, cells were washed twice in serum-free medium, were overlaid with 500 µl of serum-free ECM containing 0.2-µg/ml trypsin per well, and were maintained at 37°C for 7 days.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Isolation of IEC. Epithelial cells were digested from intestinal fragments as intact organoids (Fig. 1A). Occasionally, epithelial cell organoids were attached to fragments of small intestinal lamina propria. Following repeated gradient centrifugation, a few single cells and minimal amounts of debris remained. At least 95% of cells were viable at the time of plating as determined by trypan blue exclusion.

View larger version (54K): [in this window] [in a new window]   FIG. 1.   Phase-contrast microscopy of freshly isolated and cultured murine small IEC (A). Epithelial cells were digested from intestinal fragments as small intact organoids and stained with hematoxylin and eosin (A). Epithelial cells proliferated from the edges of the attached organoids 72 h after plating (B). After 18 days in culture, colonies of primary epithelial cells coalesced to form semiconfluent monolayers of densely packed cuboidal cells (C). Magnification, ×250 (A) and ×100 (B and C).

Growth and viability of primary small IEC in culture. Less than 5% of plated epithelial cell organoids attached to culture surfaces and generated colonies of new cells. Attached organoids spread out onto culture surfaces within 24 h; by 48 h after plating, new cells were visible, migrating out from the edges of organoids as cohesive monolayers with distinct margins (Fig. 1B). Cells exhibited typical epithelial cell morphology and developed into compact cobblestoned monolayers. Proliferating colonies of IEC coalesced to form large confluent areas of cells (Fig. 1C). Nonepithelial cells had a smooth-muscle-cell-like appearance and were present infrequently, at the edges of the IEC colonies or at the periphery of the well. We found that culture medium containing less than or equal to 2% FBS encouraged proliferation of IEC and inhibited the growth of mesenchymal cells (data not shown). Three to four weeks after plating, cultured IEC began to degenerate, monolayers lost their compact appearance, and cells died. If culture surfaces were not precoated with Matrigel, attachment and proliferation of cells in the first 48 h occurred. However, cells rapidly degenerated approximately 4 to 5 days after plating.

Immunohistochemical characterization of primary epithelial cells. (i) Freshly isolated epithelial cells. Greater than 95% of freshly isolated cells expressed cytokeratin (Fig. 2A). Freshly isolated epithelial cells also expressed the intestinal disaccharidases lactase and sucrase (Table 2) and alkaline phosphatase (data not shown). In contrast, vimentin (found in fibroblasts and neural cells) and -smooth-muscle actin (found in smooth-muscle cells) were detected in less than 5% of cells obtained prior to plating (data not shown).

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Cytokeratin was detected in freshly isolated organoids of epithelium (A) and cultured IEC grown for 4 days on coverslips (B). Cells were fixed in 1% paraformaldehyde and were stained with rabbit anti-broad spectrum cytokeratin, followed by a mouse anti-rabbit FITC-labeled antibody. Intestinal alkaline phosphatase activity was detected in cultured IEC grown on coverslips for 7 days by using the Vector Red substrate kit with the addition of levamisole (C). Immunofluorescent detection of rotavirus antigen in primary cultured small IEC from suckling mice was carried out 72 h after infection with RRV at an MOI of 1. Cells were fixed in 1% paraformaldehyde and were stained with polyclonal rabbit anti-WC3 rotavirus antibody, followed by mouse anti-rabbit FITC-labeled antibody. Magnification, ×1,000 (A and B) and ×250 (C and D).

(ii) Cultured epithelial cells. Immunohistochemical characterization of cultured epithelial cells revealed that greater than 99% of cuboidal cells expressed cytokeratin 2 to 19 days after plating (Fig. 2B); the intensity of cytokeratin expression did not diminish during this time. The majority of nonepithelial cells in primary cultures were smooth-muscle cells, as determined by the expression of -smooth-muscle actin. Smooth-muscle cells usually represented less than 10% of cells in culture and were detected either at the periphery or underlying the colonies of IEC. Vimentin was expressed in less than 1% of cells (data not shown). Primary IEC did not express vimentin or -smooth-muscle actin after 19 days in culture. Expression of intestinal alkaline phosphatase diminished rapidly in cultured IEC during the first 3 days after plating. However, low-level expression was detected for 14 days in culture (Fig. 2C). Disaccharidase expression was not detected in cultured IEC assayed between 6 and 19 days after plating and was also not detected in m-IC_cl2 cells grown between passages 50 and 60 on rat tail collagen (Table 2).

Class II antigen expression by cultured murine IEC. Since expression of class II antigens is age dependent and not detectable until at least 7 days after birth (5, 26), we chose to examine Ia^d expression in cells cultured from adult mice in addition to cells cultured from suckling animals. Freshly isolated epithelial cells from suckling mice had less than 3% expression of Ia^d, whereas Ia^d was detected in 23% of freshly isolated epithelial cells from adult BALB/c mice (data not shown). Ia^d continued to be expressed constitutively in 19% of cultured cells (25% of epithelial cells) from adult mice, obtained 48 h after plating (Table 3).

Rotavirus infection of murine small IEC. (i) RRV. Primary cultured epithelial cells inoculated with RRV at an MOI of 1 showed cytopathic effect (CPE) in less than 5% of cells at 24 h, but CPE increased to involve approximately 5 to 10% of cells at 72 h after infection (data not shown). Cells at the margins of the IEC colonies were condensed and clustered, and the number of dead cells floating in the medium increased. CPE was not observed in m-IC_cl2 cells after infection with RRV (data not shown). Rotavirus antigen was detected by immunofluorescence assay in RRV-infected primary IEC cultures 72 h after infection in at least a 125-fold greater quantity than in m-IC_cl2 cells (Fig. 2D and Table 4). Amplification of virus above the input inoculum was approximately 40-fold for primary IEC but was not observed for m-IC_cl2 cells (Table 5). Quantities of infectious RRV detected in primary IEC cultures were approximately 130-fold greater than those detected in m-IC_cl2 cells.

(ii) Cell-culture-adapted EDIM rotavirus. CPE was not observed in primary IEC or m-IC_cl2 cells inoculated with a cell-culture-adapted strain of EDIM (data not shown). Cell-culture-adapted EDIM antigen was present in a greater quantity in primary IEC cultures than in m-IC_cl2 cells (Table 4). However, fewer fluorescent foci were seen in primary IEC infected with cell-culture-adapted EDIM than in primary cultures infected with RRV (Table 4).

(iii) Wild-type EDIM rotavirus. Wild-type EDIM intestinal homogenate and control intestinal homogenate produced cytotoxic changes in both cell types to an equal degree; no virus-specific CPE was observed (data not shown). Viral antigen was not detected by immunofluorescence assay in cultures of either primary IEC or m-IC_cl2 cells treated with wild-type EDIM virus (Table 4).

	DISCUSSION

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We developed a method for long-term culture of primary murine small IEC. In previous studies, small IEC have been difficult to maintain in culture, remaining viable for only hours to several days (for review, see reference 15). Although long-term cultures of human and rat small IEC have been established (25, 27), IEC derived from mice have depended on immortalization by simian virus 40 transfection to remain viable (4, 31, 33). Successful cultivation of small IEC in this study was dependent on a number of factors. First, digestion of intestinal epithelium by using collagenase and dispase elaborated viable intact organoids of epithelium (7, 10). Plating organoids, some presumably from crypts containing viable stem cells, was essential to generate proliferating epithelial cells in culture. Efforts to culture IEC from single-cell suspensions were unsuccessful (data not shown). Second, the presence of a limited number of nonepithelial cell types in the cultures (predominantly smooth-muscle cells) may have also contributed to IEC growth and viability (7, 10, 29). We found that overgrowth of cultures with mesenchymal cells occurred at concentrations of FBS greater than 2.5%. Epithelial cell growth was enhanced in culture medium containing less than or equal to 2% FBS (10, 27). Third, coating culture surfaces with Matrigel (a basement membrane matrix) was important in maintaining long-term cultures of IEC (3, 27, 29). Matrigel may induce differentiation of epithelial cells while reducing longevity of cells in culture (19, 27, 29). Fourth, the synergistic activity of growth factors such as epidermal growth factor, insulin, dexamethasone, selenium, triiodothyronine, and transferrin also likely contributed to sustained growth of IEC (15).

Primary cultured epithelial cells retained morphologic and functional characteristics of relatively immature small IEC. Morphologically, IEC proliferated from attached organoids as confluent sheets of cuboidal cells. The presence of cytokeratin, an intermediate cytoskeletal filament that traverses the cell cytoplasm, is characteristic of epithelial cells (21). Previous studies showed that expression of cytokeratin in cultured IEC diminishes over time (6, 33). In addition, primary epithelial cells may convert to a mesenchymal cell phenotype (as determined by the expression of vimentin) in long-term culture (15). We found that cultured primary IEC expressed cytokeratin for 3 weeks (with an intensity comparable to freshly isolated epithelial cells) and did not acquire the characteristics of other cell types. Primary IEC also continued to express the intestinal isoenzyme alkaline phosphatase throughout the culture period, although expression was reduced relative to that of freshly isolated cells. Intestinal disaccharidases (particularly lactase and sucrase) were expressed by freshly isolated IEC, but not by cells maintained in culture for 6 to 19 days. Assay for disaccharidases at earlier time points in culture was precluded by the difficulty in obtaining adequate numbers of cells.

We found that approximately 23% of freshly isolated small IEC from adult mice expressed class II antigens and that expression was maintained in culture for 48 h. MHC class II molecules are known to be constitutively expressed in small IEC and are localized on the basolateral surface of cells lining the upper two-thirds of the villus (5). Class II molecules are absent on intestinal epithelium during fetal development; however, expression may be apparent as early as 7 days after birth, presumably in response to stimulation by nonself antigens (5, 26). Consistent with these findings, we found minimal expression of Ia^d on both freshly isolated and cultured IEC from 7-day-old mice. It is unclear whether IEC bearing class II antigens participate in rotavirus-specific responses; however, murine small IEC bearing class II antigens have been shown to present nominal antigens to primed T cells (14, 32). We are currently conducting studies in our laboratory to determine if primary IEC can act as rotavirus-specific antigen-presenting cells.

Achieving differentiation in vitro of nontransformed cultured IEC to functionally mature enterocytes has been elusive, in both this and previous studies (15). Progress in optimizing culture conditions has been limited because factors that govern differentiation of stem cells to mature disaccharidase-producing epithelial cells in vivo have not been fully determined. However, although rotaviruses infect differentiated enterocytes of the intestinal villi in vivo, lack of cell differentiation in vitro may have less effect on the susceptibility of cells to rotavirus infection. For example, both differentiated and undifferentiated Caco-2 cells are susceptible to rotavirus infection with either simian strain RRV (1, 12) or human rotavirus strain Wa (12). In this study, although primary cultured IEC did not differentiate to mature enterocytes, these cells were highly susceptible to rotavirus infection as compared with the murine small intestinal cell line, m-IC_cl2.

Since much of our understanding of rotavirus pathology and immunology has been derived from a murine model of rotavirus infection, using both heterologous and homologous rotavirus strains (11, 18, 20, 22, 23), the susceptibility of primary cultured IEC to different strains of rotavirus was determined. Primary-cultured small IEC from suckling mice supported the growth of RRV to a significantly greater extent than m-IC_cl2 cells. The yield of infectious RRV from primary cultured epithelial cells was maximal at 24 h and a CPE was greatest 72 h after infection. This is consistent with studies of rotavirus in other cultured cell types demonstrating production of infectious virus prior to the appearance of a maximal CPE (13, 23). The proportion of cultured IEC infected with RRV was approximately 5 to 10% by immunofluorescence assay, comparable with previous in vivo studies of simian rotavirus strain SA11 in adult mice, in which 5 to 10% of villus epithelial cells contained rotavirus antigen (23). Although rotavirus-specific immunofluorescence was detected in primary-cultured IEC after infection with cell-culture-adapted EDIM, the number of stained cells was significantly less than that found after infection with RRV. Wild-type murine rotavirus EDIM was not detected in either cell type by immunofluorescence assay.

The limited susceptibility of cultured murine IEC to wild-type or cell-culture-adapted EDIM may have been due to several factors. First, lower quantities of infectious virus are likely present in wild-type EDIM preparations, as compared with preparations of RRV. Second, although 7-day-old mice were used because of the age-dependent susceptibility to rotavirus infection in young animals, the chronological age of the cultured IEC was at least 14 days postnatal by the time of infection, a factor that may have contributed to the lack of viral infectivity. Third, the absence of numerous other factors present in vivo at the intestinal mucosal surface that may be required for viral attachment and entry (such as colonizing bacteria, intestinal enzymes, glycocalyx, and milk components) likely contributes to the difficulty of adapting murine viruses to growth in vitro. The susceptibility of primary-cultured IEC to cell-culture-adapted EDIM suggests that this virus, although adapted to growth in vitro in MA104 cells, could be adapted to growth in primary murine IEC by serial passage. We are currently undertaking these studies in our laboratory.

Although organ culture has been employed to a limited extent in the study of rotavirus pathogenesis (2, 28), this study is, to our knowledge, the first to describe rotavirus infection of primary-cultured murine IEC. Optimal infection of cultured cells by rotavirus requires cleavage of the outer capsid protein vp4 by trypsin (8). The ability to maintain cultured primary epithelial cells for several days in serum-free culture medium containing trypsin enabled productive rotavirus infection of IEC. Long-term primary culture of murine small IEC provides a method to facilitate studies of rotavirus-epithelial cell interactions in vitro and to explore the capacity of IEC to act as antigen-presenting cells for rotavirus in an MHC-compatible system.