Rotavirus infection impairs intestinal brush-border membrane Na+-solute cotransport activities in young rabbits

Nabil Halaihel, Vanessa Liévin, Francisco Alvarado, and Monique Vasseur

Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na+-D-glucose (SGLT1) and Na+-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na+-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established.

viral diarrhea; rabbit intestine; SGLT1; sodium-solute symport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VIRAL DIARRHEAS ARE THE CAUSE of high mortality among children and young animals. However, in spite of a great deal of research over several decades, the mechanism(s) underlying these diarrheas remains unclear.

Rotavirus and coronavirus (the transmissible gastroenteritis virus), two entirely distinct viruses, exhibit similar tropism for the epithelial cells at the tips of the small intestinal villi (for review, see Ref. 17). Both viruses can cause extensive enterocyte losses, leading to flattening of the mucosa, and induce a watery diarrhea that can be fatal if incorrectly treated (17, 23, 30, 33, 34). However, whether or not diarrhea is the result of malabsorption after tissue damage is open to question. Indeed, the idea is gaining ground that diarrhea is not necessarily a consequence of any physical lesion but can precede it, as if cell dysfunction were the cause rather than the consequence of the histological damage (2, 4, 7, 31, 38).

Further analogy between rotavirus and coronavirus includes the fact that both cause a reduced capacity for intestinal NaCl and nonelectrolyte absorption (9, 17, 22, 23, 28). Both D-glucose and L-alanine absorption are impaired, although that of L-alanine appears to be affected only partially (9, 28). By using brush-border membrane (BBM) vesicles isolated from the jejunum of 14-day-old piglets experimentally infected with coronavirus, Keljo and colleagues (22) showed the existence of two kinetically distinct D-glucose transport systems, one of which was selectively abolished after coronavirus infection. The inhibited system was the well-known, high-affinity Na+-D-glucose symporter, presently identified as SGLT1 (20).

Because the Na+-D-glucose symporter was suspected to be present in the BBM of the mature enterocyte but absent from the crypt cells, Hamilton and colleagues (9, 17, 22, 23, 28) proposed that virus infection kills off most of the mature enterocytes, so that crypt cells invade the villus surface, rendering it unsuitable for absorption. The resulting malabsorption would be the direct cause of the diarrhea.

This crypt-cell invasion hypothesis, however, has never been conclusively demonstrated, and in fact has been challenged by more recent work from other laboratories (8, 30). The present study concerns an alternative explanation for the mechanism of viral diarrhea, compatible with the original observation by Keljo and colleagues (22) that viral infection selectively and strongly inhibits SGLT1 activity.

The existence of a mechanistic relation between the pathogenesis of viral diarrhea and impaired Na+-coupled D-glucose transport becomes apparent when comparing these diarrheas with the human genetic disease D-glucose and D-galactose malabsorption. The main symptom of this disease is a watery diarrhea unequivocally attributable to the absence of SGLT1 in the intestine of babies lacking the corresponding gene (41). Because both glucose-galactose malabsorption and the above-mentioned viral diarrheas have in common a watery diarrhea accompanying either a genetic or an acquired dysfunction of SGLT1, in the present study we have sought to establish whether or not protein expression and activity of SGLT1 are both similarly impaired in the course of rotavirus infection. An effect on SGLT1 activity, unaccompanied by any effect on SGLT1 protein expression, would constitute compelling evidence that an alternative to the crypt cell invasion hypothesis is necessary.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Rotavirus source. Two 7-wk-old New Zealand rabbits at the Bergerie Nationale (stock farm) (Rambouillet, France) were identified as diarrheic. Considered to be naturally infected, presumably by rotavirus, the animals were brought to our laboratory and immediately killed by cervical dislocation after stunning. Samples of the intestinal fluid were filtered through a 0.45-µm membrane (Analypore, OSI) and used for virus identification and multiplication in MA-104 monkey kidney cells in culture (21). By using material from one animal (LN2; see below) and three passages in MA-104 cells, a rotavirus stock suspension was thus obtained. Named rotavirus strain "La/RR510" or "RR510" (the lapine rotavirus, strain La/RR510, has been deposited into the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France), it is stored at -80°C. It has been used as the source of rotavirus for all the studies that follow.

Virus identification and quantitation. Viral RNA was extracted directly from the intestinal fluid of both naturally and experimentally infected rabbits by using a (1:1) phenol-chloroform mixture in the presence of 1% SDS. Electrophoretic analyses were performed as described previously (36). RNA bands were stained with the Bio-Rad Silver Stain Plus kit. Rotavirus present in the intestinal fluid of experimentally infected rabbits was quantitated by enzyme immunoassay (IDEIA rotavirus test, DAKO Diagnostics) and by titration on MA-104 cell monolayers after a complete cytopathic effect was obtained (21). The titer in infectious particles per milliliter (ip/ml) was calculated according to the method of Wyshak and Detre (42). Virus was also quantitated in MA-104 cell cultures by immunofluorescent assay (21).

Identification of rotaviral proteins in both intestinal homogenates and purified intestinal BBM vesicles was done by Western immunoblot analysis. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels, electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad), and immunostained as described previously (24). The rabbit polyclonal antirotavirus antibody used (no. 8148) was the gift of Dr. J. Cohen of INRA (Jouy-en-Josas, France). It was used at a dilution of 1:5,000.

The presence of intact rotavirus particles in the purified BBM vesicle preparations was demonstrated by electron microscopy after negative staining, as described by Zeng et al. (43). All micrographs were taken with a Philips EM-208 electron microscope operating at 80 KV.

Rabbit inoculations and sample collection. Specific pathogen-free (SPF) albino hybrid New Zealand 4- or 7-wk-old rabbits were obtained from Charles River. Animals were maintained in positively pressurized rooms at a temperature of 18-20°C at the animal house of the University of Paris-Sud (Châtenay-Malabry, France). All animals received a commercial diet (112 UAR, Villemoisson-sur-Orge, France) and water ad libitum. Rabbits were orally inoculated with 2 ml of the rotavirus stock suspension (105 ip/ml) by means of a pediatric feeding tube.

Fecal samples were collected daily and scored for consistency as either normal, pasty, or diarrheic. Diarrhea is defined as characterized by fluid stools accompanied by fecal staining of the perineum. At appropriate times after infection, animals were killed, and for each animal the entire contents of the small intestine were collected for virus detection and titration. Both jejunal and ileal samples were taken for histological examination after fixing in 10% formalin at room temperature. Longitudinal and transversal sections (4 µm) of paraffin-embedded tissue were stained with Mayer's hematoxylin and eosin. Crypt height and villus depth were determined in a blinded fashion as described previously (25). For transport study, either jejunal or ileal segments from 7-wk-old rabbits or the entire small intestine from 4-wk-old rabbits were removed, rinsed with saline at room temperature, everted, and distributed into plastic bags for storage at -80°C, as described previously (37).

Intestinal BBM vesicle preparation. Intestinal BBM vesicles were prepared by using frozen intestine and the magnesium precipitation method as described previously (18). After suspension at ~40 mg protein/ml in the buffer described below, vesicles were stored in aliquots of 200 µl in liquid nitrogen until the day of the transport assay, as described previously (37). The membrane buffer consisted of 20 mM HEPES and 10 mM Tris · HCl, supplemented with D-arabinose to a total of 600 mosM and adjusted to pH 7.4 with Tris base (37). Viral and membrane protein concentrations were determined by using the Bio-Rad protein assay kit, with serum globulin as standard.

Transport assays. Transport was assayed by using a rapid filtration technique (6) and either D-[14C]glucose or L-[14C]leucine as the substrate. Briefly, 10 µl of a BBM vesicle preparation were used to carry out uptake measurements by mixing with 40 µl of transport buffer formed by the membrane buffer containing variable concentrations of unlabeled substrate (0.1-150 mM), 14C-labeled substrate as a tracer, and 100 mM NaSCN (final concentrations in the incubation mixtures). Initial uptake rate measurements were then carried out for 2.6 s at 35°C (6). After background subtraction (16), uptake results were calculated as absolute velocities (in pmol · s-1 · mg membrane protein-1) ± SD. Data were statistically compared by applying a global one-way ANOVA (32).

Kinetic analyses. Uncorrected, initial absolute entry rates as a function of the substrate concentration ([S]) were fitted by iteration to an equation involving the sum of one nonsaturable, diffusion-like component and one Michaelian, saturable transport component:
v<IT>=</IT>{[<IT>V</IT><SUB>max</SUB><IT>/</IT>(<IT>K</IT><SUB>T</SUB><IT>+</IT>[S])]<IT>+K</IT><SUB>d</SUB>}<IT>·</IT>[S]
where Vmax (maximum velocity) and KT (the apparent Michaelis constant) are the capacity and affinity parameters of classical Michaelis-Menten kinetics, respectively, and Kd is an apparent diffusion constant (6, 16). To perform each fit, the procedure of Fletcher and Powell as modified by van Melle and Robinson (39) was used. Using the commercial program Stata (Integral Software, Paris, France), we fitted the nonlinear least-squares regression functions in a single run to each data set by minimizing the sum of squares of errors. By comparing "lack of fit" and "pure error" components, we obtained F values providing a quantitative assessment of the goodness of fit (39). All of the fits listed in the tables were found to be not significant (see Ref. 39), meaning that, for each given fit, data points did not differ statistically from the theoretical fit of the equation under study. Statistical comparison between different fits was done by applying the F'test of van Melle and Robinson (39). All calculations were done using Apple Macintosh microcomputers.

Phlorizin binding measurements. Quantitation of the SGLT1 protein present in the isolated BBM vesicles was performed by measuring phlorizin binding as described by Garriga et al. (13). The density of specific phlorizin binding sites was computed as the difference between binding in the presence of either NaCl or KCl and is expressed as picomoles bound per milligram of membrane protein at a 50 µM phlorizin concentration (B50).

SGLT1 protein identification in isolated BBM vesicles. BBM proteins (60 µg/well) were separated by SDS-PAGE on 8% polyacrylamide gels and immunoblotted as described previously (14). We used a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 564-575 of the adult rabbit SGLT1 sequence (14). The antibody, used at 1:5,000 dilution, was a gift of Dr. M. Kasahara of Teikyo University (Tokyo, Japan).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of group A rotavirus in intestinal contents of naturally infected rabbits. Two young adult, 7-wk-old, naturally infected rabbits, identified because of their having diarrhea, were used to investigate the presence of rotavirus in their intestinal contents. A double-stranded RNA was found, exhibiting a series of 11 bands with a characteristic 4, 2, 3, 2 pattern (results not shown), which indicated the presence of a typical group A rotavirus. Further analyses, including electron microscopy, MA-104 cell-culture immunofluorescent assay, IDEIA rotavirus test, and immunoblot analysis, confirmed that a group A rotavirus was present (Fig. 1).


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Fig. 1.   Identification of RR510 rotaviral proteins and particles in intestinal homogenates and brush-border membrane (BBM) vesicles isolated from the naturally infected rabbit LN1 (see Table 1). A: Western immunoblot analysis of the jejunal homogenate (lane a), purified jejunal BBM vesicles (lane b), purified ileal BBM vesicles (lane c), RR510 virus stock (lane d), and jejunal homogenate (lane e) with jejunal BBM vesicles from 1 control, healthy rabbit of the same age (lane f). Horizontal arrows indicate position of the molecular mass markers. B: negative staining electron micrograph of the jejunal BBM vesicle preparation shown in lane b. Magnification bar, 100 nm.

On necropsy, a net increase in intestinal fluid volume was observed in the naturally infected animals, compared with that in healthy rabbits of the same age. Significant histological lesions were seen in both the jejunum and ileum of the naturally infected animal LN2 (Fig. 2). The lamina propria exhibited a moderate, polymorphous inflammatory state, and there was villus atrophy, with crypt length-to-villi length ratios (0.34 ± 0.08, n = 2) significantly increased compared with the controls (0.13 ± 0.03, n = 8). However, it should be noted that even when, in this animal, slight erosion of the epithelial surface existed, there was no evidence of any significant enterocyte loss or flattening of the mucosa (Fig. 2).


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Fig. 2.   Representative photomicrographs from the jejunum of 7-wk-old rabbits (see Table 1). Tissues were stained with hematoxylin and eosin. Magnification, ×300. A: healthy control. B: infected animal LN2.

To study rotavirus infection effects on transport, BBM vesicles were isolated from the frozen jejunum and ileum of both healthy and infected rabbits. All the vesicles were normal (results not shown) according to a series of previously defined criteria (37). Interestingly, all BBM vesicles purified from infected, 7-wk-old rabbits contained RR510 antigens and particles, as determined, respectively, by Western immunoblot analysis and electron microscopy (see Fig. 1). Because VP7 was among the proteins detected, it can be concluded that intact triple-layered particles were present, although the data available do not permit establishing whether or not double-layered particles were also present.

Quantitation of RR510 infection in experimentally infected rabbits. In a preliminary experiment, two SPF rabbits of the same age as that of the field animals (7 wk) were inoculated with 2 ml of the RR510 virus stock, a dose found to effectively cause virus replication in these animals (see below for more detail). However, the animals did not develop diarrhea and were all killed at 72 h postinfection (hpi). It was found that, in contrast with the naturally infected rabbit, no histological damage had occurred, as evinced by the ratios of crypt length to villi length (0.16 ± 0.03, n = 8), which were indistinguishable from those of the controls.

In a second series of experiments, 14 SPF 4-wk-old animals were used to see if tissue damage and/or diarrhea would be manifested at this age. After inoculation with the same dose of RR510 virus used above, groups of three animals were killed at the time intervals shown in Fig. 3. As indicated in Fig. 3, similar to the 7-wk-old animals, feces had a normal consistency throughout the first 72 h in 4-wk-old animals (5 of 5 animals). However, a transient, mild, watery diarrhea appeared at 124 hpi, replaced by pasty feces at 144 hpi. Rotavirus could be detected in the intestinal lumen (IDEIA rotavirus test and titration) of each of the infected animals from the earliest time examined (16 hpi) onward. At 40 and 72 hpi, the excreted viral particles exceeded the concentration of the input dose by >12- and 600-fold, respectively. They then dropped to <4 × 102 ip/ml by 144 hpi (Fig. 3). It should be emphasized that, in spite of the large amounts of virus produced, none of the intestinal samples examined during this period exhibited any apparent histological damage. The crypt length-to-villi length ratios for control and infected animals were statistically identical up to 144 hpi (Fig. 3).


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Fig. 3.   Time-dependent viral shedding and characteristics of feces and intestine of 4-wk-old rabbits infected experimentally with RR510 rotavirus. Rotavirus excreted in the intestinal fluid was quantitated by either IDEIA rotavirus test [, expressed as optical density (OD) at 450 nm] or titration on MA-104 cell monolayers [black-triangle, given as the logarithm of infectious particles/ml (ip/ml)]. a: the consistency of feces where N = normal, D = diarrheic, and P = pasty. b: crypt-to-villi length (C/V) ratios where the results were statistically indistinguishable from one another (P < 0.01). c: group A rotavirus detected by electrophoretic analyses in intestinal fluid; - and + indicate negative and positive results, respectively. d: rotavirus antigens and particles detected in isolated BBM vesicles (BBMVs) by immunoblot and electron microscopy, respectively.

Concerning the identity of the virus shed into the intestinal fluid of all experimentally infected rabbits (RNA electropherotype) independently of age, it was indistinguishable from that originally isolated from the field animals. Rotaviral proteins (immunoblot analysis) were detected in BBM vesicles of either all naturally and experimentally infected, 7-wk-old rabbits or the 4-wk-old rabbits, although here this result was measurable only at 72 hpi (Fig. 3).

Effect of rotavirus infection on kinetics of D-glucose and L-leucine uptake. To establish whether or not intestinal Na+-organic solute symport activities are affected in the course of rotavirus enteritis, D-glucose and L-leucine saturation curves were performed by using BBM vesicles from either normal or infected rabbits. With both 4- and 7-wk-old animals, a strong inhibition of the Na+-coupled transport of each D-glucose and L-leucine was apparent in infected rabbits, independent of whether the infection occurred naturally or experimentally (Tables 1 and 2).

                              
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Table 1.   Kinetics of D-glucose and L-leucine uptake by BBM vesicles purified from naturally or experimentally infected, 7-wk-old rabbits


                              
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Table 2.   Evolution with time of the kinetics of D-glucose and L-leucine uptake by intestinal BBM vesicles purified from experimentally infected, 4-wk-old rabbits

For both symports, Vmax was the only parameter affected. In contrast, both the KT and the Kd parameters remained practically unchanged under all conditions studied. As shown in Tables 1 and 2, all KT values fell within the normal limits generally found for this parameter in regular intestinal transport studies, where KT is often found to vary by factors between 1 and 2, occasionally more. Concerning Kd, independent of age, all the D-glucose (Tables 1 and 2) and L-leucine (Table 1) results were essentially identical. One set of Kd results (L-leucine, Table 2) appeared to be double the rest, but this difference can in principle be attributed to the use of a different batch of filters in this particular experiment. It is known that different filter batches can cause Kd measurements to change slightly but significantly (e.g., see Ref. 27). However, this small filter effect can be neglected, particularly since it has no significant impact on the estimation of the critical Michaelian kinetic parameters, Vmax and KT. As demonstrated by the stability of the Kd parameter within each given set of experiments (Tables 1 and 2), the integrity of the BBM vesicles appears not to have been affected, even at times as long as 144 hpi, a result indicating that rotavirus infection does not modify the stability of the membrane. Nevertheless, to verify this crucial point, additional experiments were performed to measure the apparent vesicular volume, which, as defined by Brot-Laroche and Alvarado (5), can be interpreted as a measure of the functional vesicle yield. In this separate set of experiments, equilibrium uptakes were determined after vesicle incubation for 90 min at 22°C. It was found that, under all conditions studied, this parameter remained constant at 0.6 ± 0.1 µl/mg protein (n = 11), confirming that the integrity and yield of BBM vesicles did not change in the course of rotavirus infection.

The entire set of kinetic results obtained (Tables 1 and 2) indicates quite clearly that Vmax is the only parameter systematically affected by rotavirus infection. With the 7-wk-old animals, there was a certain degree of scattering in the Vmax results (Table 1), but this occurred at random and can be attributed to animal variability. Thus, for instance, by applying the F'test and comparing individual animal results by pairs, the animals studied could be separated into two groups (see Table 1 and Fig. 4). One group was formed by animals LN1 and LE2 (overall fit 1), exhibiting an identical Vmax inhibition of 70%. The other group was formed by animals LN2 and LE1 (overall fit 2), exhibiting a significantly different inhibition of 91%. The statistical value of this series of experiments is strengthened by the fact that no significant difference was found between jejunal and ileal BBM vesicles isolated from the same given animal. This is why the relevant results have been pooled and are shown as overall fits, jejunum and ileum, in Table 1 and Fig. 4. Incidentally, this result confirms the existence of a kinetic homogeneity of sugar transport activities along both of these segments of the small intestine (29). Another conclusion of interest is that the observed variability in the Vmax results takes place irrespective of any difference between natural and experimental infection, as well as between the presence or absence of intestinal lesions. It can be attributed exclusively to variability between animals within this age group (7 wk). We have no explanation for the observed difference between animals, but, in any case, these differences do not affect in the least the general conclusion reached in this study, that Vmax is the only kinetic parameter affected by rotavirus infection.


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Fig. 4.   D-glucose uptake by intestinal BBM vesicles purified from naturally and experimentally infected 7-wk-old rabbits. Saturation curves were performed under standard conditions in presence of an out/in = 100/0 mM NaSCN gradient, using vesicles from either noninfected controls (), naturally infected animals LN1 () and LN2 (black-triangle), or experimentally infected animals LE1 (open circle ) and LE2 (triangle ). Results are expressed as the absolute initial D-glucose uptake rates ± SD (n = 9-11 or 4-6 determinations/point for controls and infected animals, respectively). Because no significant difference was found between animals LN1 and LE2 or between animals LN2 and LE1, the corresponding results have been pooled by pairs and fitted again to obtain, respectively, the overall fits 1 and 2 (see Table 1). From here were then computed the theoretical fits illustrated by the lowest two curves. All fits were performed by using whole sets of available results from 0.1 to 150 mM substrate concentration, but only the data from 0.1 to 10 mM D-glucose are depicted.

Because of the small size of 4-wk-old rabbits, transport assays were performed here by using BBM vesicles isolated from the entire small intestine of these animals. As depicted in Fig. 5, the inhibition of Na+-D-glucose symport increased as a function of the time after infection and indicated the existence of a selective, progressive fall in the Vmax parameter, which dropped by ~45% in 16 h and 83% at 144 hpi (Table 2). The different groups of infected animals were all statistically different from the control group. In contrast with the 7-wk-old animals (Table 1), there were no significant differences between individual animals within each given time period.


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Fig. 5.   D-glucose uptake by intestinal BBM vesicles purified from experimentally infected 4-wk-old rabbits. Saturation curves were performed under standard conditions in presence of an out/in = 100/0 mM NaSCN gradient, using vesicles from either noninfected controls () or infected animals at 16 (), 72 (black-triangle), or 144 (diamond ) h postinfection (hpi). Results are expressed as the absolute initial D-glucose uptake rates ± SD (n = 12-15 determinations/point). Solid lines represent theoretical fits computed by using the equation given in METHODS and the kinetic parameters listed in Table 2. As in Fig 4, only the data from 0.1 to 10 mM D-glucose have been depicted.

Concerning the Na+-L-leucine symporter, the Vmax parameter also decreased as a function of the time after infection, but significant inhibition was evident only at 72 h, compared with 16 h for D-glucose. There seems to be a parallelism in the Vmax parameter decay observed for either D-glucose or L-leucine after rotavirus infection at long time periods (1 to 6 days). However, a quantitative analysis of the Vmax results (not illustrated graphically) revealed important differences. The L-leucine results followed a single exponential with a decay time constant of 0.22 day-1. In contrast, the D-glucose data involved two exponentials, namely, a rapid one with a decay time constant of 0.91 day-1 plus a second one equal to that characterizing L-leucine (0.22 day-1). We conclude that rotavirus infection causes a strong inhibition of the Vmax parameters respectively characterizing the symport with Na+ of both D-glucose and L-leucine. Both of these inhibitions took place in the absence of any apparent histological damage.

We have also investigated whether the Vmax effect was due to a rotavirus-induced drop in the size of the NaSCN gradient responsible for uphill D-glucose transport. The results shown in Fig. 6 reveal the existence of clear overshoots, as demonstrated by the 60 s/equilibrium D-glucose concentration ratios, r60, that are greater than unity, even at times as long as 144 hpi (namely, r60 = 1.4). The size of these overshoots diminishes with the time after infection, indicating that the rate of substrate influx was inhibited but the size of the underlying Na+ gradient was not affected by the infection. This is the result to be expected if rotavirus infection selectively impairs the turnover rate of the symporter.


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Fig. 6.   Time course of D-glucose uptake into intestinal BBM vesicles purified from experimentally infected 4-wk-old rabbits. The uptake of 0.1 mM D-glucose was measured at 22°C in presence of an out/in = 100/0 mM NaSCN gradient, using vesicles from either noninfected controls () or infected animals at either 16 (), 40 (open circle ), 72 (black-triangle), or 144 (triangle ) hpi. Results are expressed as the absolute uptakes, with n = 3 determinations/point. SD is smaller than the symbols. Inset: the scale has been reduced to better illustrate the difference between the lowest 2 curves.

Finally, to detect possible changes in the total mass of the Na+-D-glucose symporter per unit weight of membrane protein, specific phlorizin binding and the amount of SGLT1 antigen present in the purified BBM vesicles were investigated. First, phlorizin binding was found not to change as a function of time after rotavirus infection, the B50 values remaining constant at 39 ± 6 pmol/mg membrane protein (n = 10), independent of time. Second, as determined by densitometry, the amount of SGLT1 antigen present in the purified BBM vesicles was also found to be practically the same at 144 hpi and in the controls (relative densities of 125 and 100, respectively; Fig. 7). It is worth emphasizing that the signal given by the young rabbit BBM vesicles exhibited a single band with a molecular mass of 75 kDa, identical to that of the adult rabbit (14).


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Fig. 7.   Western blot identification of the SGLT1 antigen in intestinal BBM vesicles from 4-wk-old rabbits. Lane a: noninfected controls; lane b: infected animals at 144 hpi. Horizontal arrow indicates position of the molecular mass marker.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study examines whether rotavirus infection specifically impairs intestinal Na+-and-solute symport systems, which, the results suggest, may play a mechanistic role in the pathogenesis of rotavirus diarrhea. Kinetic analyses with BBM vesicles isolated from rabbits infected either naturally or experimentally with the RR510 rotavirus indicated strong inhibition of both Na+-D-glucose (SGLT1) and Na+-L-leucine symport activities. For both symporters, inhibition affected only the Vmax parameter and no correlation was found with any intestinal lesion. In fact, in contrast with naturally infected animals, no changes in mucosal histology have been detected in any of the experimentally infected rabbits.

With 7-wk-old animals, no evidence of diarrhea occurred during the first 3 days after inoculation, which was the longest time used. Aside from the unknown length of time elapsed after infection, the presence of histological lesions only in naturally infected animals of the same age may in principle be explained in terms of the superposition of factors that are absent in the better-protected experimentally infected laboratory animals (we used SPF animals). Rotavirus disease is known to be multifactorial, and it seems logical to expect that the symptoms exhibited by field animals are more complex, and graver, than those observable in laboratory animals.

With 4-wk-old rabbits, mild diarrhea did occur, but only after 124 hpi. Even when the symptomatology was mild, it is clear that, in all of our experiments, the animals were successfully infected, as indicated by the fact that the virus titer in the intestinal lumen strongly increased with time (Fig. 3). The possibility exists that the virus had been attenuated during its passage through the MA104 cell-culture multiplication procedure (see Refs. 4, 7, and 38). The fact that the progressive inhibition of both the Na+-D-glucose and Na+-L-leucine symporters took place in the absence of any apparent histological damage, up to at least 6 days after infection, suggests that these inhibitions can be important, although not necessarily the only, determinants in the pathogenesis of rotavirus diarrhea.

Although diarrhea is characteristic of rotavirus enteritis, it is not an obligatory symptom (e.g., see Ref. 7). Appearance of clear-cut diarrhea varies widely among animals, depending on factors that include the animal species, age, and virus strain. Although severe or moderate diarrhea has been found to occur occasionally in rabbits infected with homologous rotavirus strains (35), virus shedding and diarrhea do not necessarily correlate (7, 12). In this study, we will apply the criterion that virulence should be evaluated in terms of the replication efficiency of the virus, not on its ability to induce the macroscopic symptom of diarrhea (12).

As shown here, rotavirus infection strongly inhibits both D-glucose and L-leucine transport into BBM vesicles from young rabbits, although the effect on amino acid transport takes place more slowly (Table 2). Such results agree with previous observations showing that, in piglets infected with either coronavirus (22, 23, 28) or rotavirus (9), both intestinal D-glucose and L-alanine transport are inhibited, although the amino acid effect is quantitatively smaller. For either symporter, Vmax was the only kinetic parameter affected.

There is only one instance in the literature (25) in which rotavirus has been found to stimulate rather than inhibit glucose and amino acid uptake. For instance, Leichus and colleagues (25) failed to find any evidence of NaCl and/or nonelectrolyte transport inhibition but instead observed an increase in short-circuit current as induced by either D-glucose or L-alanine. It is doubtful, however, that use of this particular methodological approach could explain the lack of accord between the results obtained by Leichus et al. (25) and practically all other laboratories.

Mechanism of rotavirus inhibitory effects on Na+-and-organic solute symport systems. Two main interpretations can be proposed to explain a selective rotavirus effect on the Vmax parameter. First, it can be envisaged that the absolute concentration of transporter molecules per unit mass of BBM vesicles ([CT]) drops as a result of the infection. Second, the apparent rate constant governing substrate translocation under saturating conditions (the turnover rate; K) could be the affected parameter.

An effect on [CT] could result from a drop in the number of functional vesicles as a result of the infection, for instance, due to vesicle lysis. However, this interpretation can be ruled out in view of the Kd results demonstrating that rotavirus infection neither interferes with vesicle yield nor causes vesicle lysis. Alternatively, there might be an interference with the correct insertion of newly synthesized symporters into the BBM, as has been proposed to occur, for instance, with the enzyme sucrase isomaltase in rotavirus-infected Caco-2 cells (21). At present, however, no direct information is available concerning this interesting but highly speculative proposal.

In accord with Hamilton's crypt cell invasion hypothesis (9, 17, 22, 23), a [CT] effect could result from tissue damage with substitution of the mature enterocytes by nontransporting crypt cells. Apart from the fact that, in the present study, no evidence of massive enterocyte loss has been found, this interpretation is directly negated by the following observations. First, if phlorizin binding gives a measure of the SGLT1 protein concentration present in the membrane (11, 40), our results indicate that this parameter remains constant throughout the entire duration of the animals' contact with the virus, 144 h (no data shown). Second, the concentration of SGLT1 antigen revealed in the vesicles by Western blot analysis remains constant for up to 144 hpi (see Fig. 7). Together, these results demonstrate that the total mass of SGLT1, a specific marker of the mature enterocyte population (20), is unaffected by rotavirus infection and, consequently, the observed Vmax drop cannot be attributed to any significant diminution in the size of the enterocyte population of the mucosa. Moreover, strong D-glucose transport inhibition occurs at times as short as 16 hpi, which are too short when compared with the 2-4 days that would be needed for any substantial replacement of the mature enterocyte layer by crypt cells (see Refs. 19 and 33).

We conclude that it is the K parameter that is impaired after rotavirus infection. To explain this effect, it could perhaps be proposed that, directly or indirectly, rotavirus infection modifies ion gradients across the brush border and, hence, the membrane potential, thereby causing rheogenic Na+-coupled substrate uptake to be inhibited. This possibility, however, can be rejected for several reasons. First, the short incubation times (2.6 s) used to measure the initial rates of substrate uptake are too short to permit any significant diminution in the size of the Na+ gradient to occur. Second, the transport measurements are based on use of isolated BBM vesicles in the presence of 100/0 (out/in) mM NaSCN gradient (16). Under these conditions, the membrane potential is likely to approach that imposed by the thiocyanate gradient because the permeability of this anion is ~50 times larger than that of Na+ (see Refs. 3 and 15). Even when, in the present study, the membrane potential was not measured, it seems unlikely that a rotavirus-induced increase in Na+ permeability could significantly modify this permeability ratio and, hence, the membrane potential. Third, an analysis of the D-glucose uptake rate as a function of time indicates that transport continues to occur uphill for long periods of time after infection (see Fig. 6), indicating that the Na+ electrochemical gradient energizing this uptake dissipates slowly. Such an observation agrees with published evidence (9, 17, 30) indicating that, after rotavirus infection, Na+ and Cl- fluxes are unaltered, whereas D-glucose-mediated Na+ absorption is diminished. The simplest explanation for the above-mentioned set of results is that K is the parameter selectively impaired after rotavirus infection, affecting both the D-glucose and L-leucine Vmax parameters.

In view of these facts, we conclude that either the virion itself or, most probably, some product thereof (see below for the case of NSP4), has such a direct, specific effect that K drops in the absence of any significant decrease in [CT]. A similar, direct mechanism has been recently proposed to explain the interaction of protein kinase C with rabbit intestinal SGLT1 expressed in COS-7 cells (40).

Possible role of NSP4 in pathogenesis of rotavirus diarrhea. A rotavirus nonstructural glycoprotein, NSP4, and a synthetic peptide corresponding to residues 114 to 135 [NSP4-(114-135)] both have been shown to cause age- and dose-dependent diarrhea in young rodents (2). The induction of diarrhea was specific, unaccompanied by any histological damage, and occurred within a period of ~3 h, similar to that characterizing the heat-stable toxin of Escherichia coli. Because of these analogies, it was proposed (10) that NSP4 acts as a viral enterotoxin, activating a Ca2+-dependent signal transduction pathway that alters intestinal epithelial transport. However, it is known that ionic concentrations in the stools of rotavirus- and coronavirus-infected animals, although high, are considerably less than those occurring in the secretory diarrheas caused by secretagogues such as the enterotoxins of Vibrio cholerae and E. coli (17).

Experiments now underway in our laboratory (1) show that the NSP4-(114-135) peptide has direct, in vitro inhibitory effects on the Na+-D-glucose symport activity of BBM vesicles isolated from rabbit intestine, similar to those evinced by the present in vivo study. These results (1) strongly suggest that NSP4 is at least one among other effectors directly causing SGLT1 inhibition during rotavirus infection in vivo. The participation of NSP4-(114-135) in these inhibitions may explain why the effects of rotavirus infection on transport are not instantaneous. In vitro, NSP4-(114-135) inhibits SGLT1 practically instantaneously (1), but the situation in vivo is different, probably because NSP4 is a nonstructural protein. Being absent from the mature, infective virion particle, NSP4 needs to be synthesized after infection, and time will be required for the newly synthesized protein to be released into the cytoplasm and/or the intestinal lumen and find its way to its target, the symporter molecule.

Role of specific inhibition of solute-driven water reabsorption mechanisms in pathogenesis of diarrhea. By using the Na+-D-glucose symporter, SGLT1, expressed in Xenopus oocytes, Loo et al. (26) have shown that 260 water molecules are directly coupled to each sugar molecule transported; and they have estimated that this coupling can account for the net absorption of 5 l of water/day in the intestine of normal humans. Such observations suggest strongly that the interference with water reabsorption, resulting from inhibition of the Na+-D-glucose symporter during rotavirus infection, might be a determining factor in the watery diarrheas caused by rotavirus in the absence of any histological damage to the intestinal mucosa.

The question whether, similar to that of D-glucose, L-leucine transport inhibition plays a role in the pathogenesis of rotavirus diarrhea is also worthy of consideration. It agrees with the well-known fact that organic solute, Na+, and water cotransport is a general phenomenon. Symports underlie the absorption of many nutrients, not only of glucose and amino acids (19). Consequently, it seems possible to conceive the mechanism of rotavirus diarrhea as involving a generalized inhibition of intestinal solute-dependent water reabsorption. Individual effects on the transport of several common nutrients would act additively, the result being a massive loss of water into the intestinal lumen. This massive water loss could eventually overwhelm the intestinal capacity for water reabsorption, thereby permitting the main symptom of enteritis, diarrhea, to get established.

Nevertheless, it should be recognized that the evidence provided in this study does not prove that the mechanism proposed here, inhibition of water reabsorption, explains fully the voluminous diarrhea caused by rotavirus. As mentioned, rotavirus disease is multifactorial and additional factors might be needed. We would propose that the inhibition of water reabsorption described here is a conditioning factor that paves the way for additional agents (e.g., bacterial superinfection and the enterotoxin effect of NSP4) to move in. Most probably, it is the sum of all of these elements that is responsible for the onset of full-blown diarrhea.


    ACKNOWLEDGEMENTS

We thank Dr. Joana Planas (Barcelona, Spain) for help with the detection of SGLT1 protein in the isolated brush-border membrane vesicles; Dr. Michel Lemullois (Orsay, France) for help with electron microscopy; Dr. Jean-François Flejou (Paris, France) for interpretation of the histopathologic findings; and our colleagues Catherine Linxe and Jacqueline Cotte-Laffite for assistance in the viral quantitation.


    FOOTNOTES

This work was supported in part by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Fondation pour la Recherche Médicale (Paris, France), the Association Française de Lutte contre la Mucoviscidose (AFLM), and the INCO Program of the European Economic Community (Grant ERB 3514 PL 950019).

Present address of F. Alvarado: Dpto. Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Salamanca, Salamanca, E-37007 Spain.

Address for reprint requests and other correspondence: M. Vasseur, Unité 510, INSERM, Faculté de Pharmacie, Université de Paris XI, 5, rue J.-B. Clément, 92296 Châtenay-Malabry, France (E-mail:monique.vasseur{at}cep.u-psud.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 19 July 1999; accepted in final form 3 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarado, F, Halaihel N, Ball JM, Estes MK, and Vasseur M. Rotavirus nonstructural glycoprotein NSP4 abolishes SGLT1 activity of brush border membrane vesicles from rabbit jejunum (Abstract). Z Gastroenterol 36: 326, 1998[ISI].

2.   Ball, JM, Tian P, Zeng CQ-Y, Morris AP, and Estes MK. Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272: 101-104, 1996[Abstract].

3.   Berteloot, A. Highly permeant anions and glucose uptake as an alternative for quantative generation and estimation of membrane potential differences in brush border membrane vesicles. Biochim Biophys Acta 857: 180-188, 1986[ISI][Medline].

4.   Bridger, JC, Hall GA, and Parsons KR. A study of the basis of virulence variation of bovine rotaviruses. Vet Microbiol 33: 169-174, 1992[ISI][Medline].

5.   Brot-Laroche, E, and Alvarado F. Disaccharide uptake by brush-border membrane vesicles lacking the corresponding hydrolases. Biochim Biophys Acta 775: 175-181, 1984[ISI][Medline].

6.   Brot-Laroche, E, Serrano MA, Delhomme B, and Alvarado F. Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea-pig jejunal brush border membrane vesicles. J Biol Chem 261: 6168-6176, 1986[Abstract/Free Full Text].

7.   Ciarlet, M, Gilger AG, Barone C, McAuthur M, Estes MK, and Conner ME. Rotavirus disease, but not infection and development of intestinal histopathlogical lesions, is age restricted in rabbits. Virology 251: 343-360, 1998[ISI][Medline].

8.   Collins, J, Candy DCA, Starkey WG, Spencer AJ, Osborne MP, and Stephen J. Disaccharidase activities in small intestine of rotavirus-infected suckling mice: a histochemical study. J Pediatr Gastroenterol Nutr 11: 395-403, 1990[ISI][Medline].

9.   Davidson, GP, Gall DG, Petric M, Butler DG, and Hamilton JR. Human rotavirus enteritis induced in conventional piglets. J Clin Invest 60: 1402-1409, 1977[ISI][Medline].

10.   Dong, Y, Zeng CQ, Ball JM, Estes MK, and Morris AP. The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production. Proc Natl Acad Sci USA 94: 3960-3965, 1997[Abstract/Free Full Text].

11.   Ferraris, RP, and Diamond JM. A method for measuring apical glucose transporter site density in intact intestinal mucosa by means of phlorizin binding. J Membr Biol 94: 65-75, 1986[ISI][Medline].

12.   Franco, MA, Feng N, and Greenberg HB. Molecular determinants of immunity and pathogenecity of rotavirus infection in the mouse model. J Infect Dis 174, Suppl 1: S47-S50, 1996[ISI][Medline].

13.   Garriga, C, Moreto M, and Planas JM. Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes. J Physiol (Lond) 514: 189-199, 1999[Abstract/Free Full Text].

14.   Garriga, C, Rovira N, Moreto M, and Planas JM. Expression of Na+/D-glucose cotransporter in brush-border membrane of the chicken intestine. Am J Physiol Regulatory Integrative Comp Physiol 276: R627-R631, 1999[Abstract/Free Full Text].

15.   Gunther, RD, Schell RE, and Wright EM. Ion permeability of rabbit intestinal brush border membrane vesicles. J Membr Biol 78: 119-127, 1984[ISI][Medline].

16.   Halaihel, N, Gerbaud D, Vasseur M, and Alvarado F. Heterogeneity of pig intestinal D-glucose transport systems. Am J Physiol Cell Physiol 277: C1130-C1141, 1999[Abstract/Free Full Text].

17.   Hamilton, JR. Viral enteritis. Pediatr Clin North Am 35: 89-101, 1988[ISI][Medline].

18.   Hauser, H, Howell K, Dawson RMC, and Boyer DE. Rabbit small intestinal brush border membrane preparation and lipid composition. Biochim Biophys Acta 602: 567-577, 1980[ISI][Medline].

19.   Hediger, MA, and Rhoads DB. Molecular physiology of sodium-glucose cotransporters. Physiol Rev 74: 993-1026, 1994[Free Full Text].

20.   Hwang, ES, Hirayama BA, and Wright EM. Distribution of the SGLT1 Na+/glucose cotransporter and mRNA along the crypt-villus axis of rabbit small intestine. Biochem Biophys Res Commun 181: 1208-1217, 1991[ISI][Medline].

21.   Jourdan, N, Brunet JP, Sapin C, Blais A, Cotte-Laffitte J, Forestier F, Quero AM, Trugnan G, and Servin AL. Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton. J Virol 72: 7228-7236, 1998[Abstract/Free Full Text].

22.   Keljo, DJ, Macleod RJ, Perdue MH, Burler DG, and Hamilton JR. D-Glucose transport in piglet jejunal brush-border membranes: insights from a disease model. Am J Physiol Gastrointest Liver Physiol 249: G751-G760, 1985[ISI][Medline].

23.   Kerzner, B, Kelly MH, Gall DG, Butler DG, and Hamilton JR. Transmissible gastroenteritis: sodium transport and the intestinal epithelium during the course of viral enteritis. Gastroenterology 72: 457-461, 1977[ISI][Medline].

24.   Labbé, M, Charpilienne A, Crawford SE, Estes MK, and Cohen J. Expression of rotavirus VP2 produces empty corelike particles. J Virol 65: 2946-2952, 1991[ISI][Medline].

25.   Leichus, LS, Goldhill JM, Long WH, Percy RD, Shaw V, Donovan R, and Burakoff R. Effects of rotavirus on epithelial transport in rabbit small intestine. Dig Dis Sci 39: 2202-2208, 1994[ISI][Medline].

26.   Loo, DDF, Zeuthen T, Chandy G, and Wright EM. Cotransport of water by the Na+/glucose cotransporter. Proc Natl Acad Sci USA 93: 13367-13370, 1996[Abstract/Free Full Text].

27.   Malo, C, and Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na+-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 122: 127-141, 1991[ISI][Medline].

28.   Rhoads, JM, MacLeod RJ, and Hamilton JR. Diminished brush border membrane Na-dependent L-alanine transport in acute viral enteritis in piglets. J Pediatr Gastroenterol Nutr 9: 225-231, 1989[ISI][Medline].

29.   Robinson, JWL, and van Melle G. Kinetics of the sodium/beta-methyl-D-glucoside cotransport system in the guinea pig. J Physiol (Lond) 344: 177-187, 1983[Abstract].

30.   Salim, AF, Phillips AD, Walker-Smith JA, and Farthing MJG Sequential changes in small intestinal structure and function during rotavirus infection in neonatal rats. Gut 36: 231-238, 1995[Abstract].

31.   Shaw, RD, Hempson SJ, and Mackow ER. Rotavirus diarrhea is caused by nonreplicating viral particles. J Virol 69: 5946-5950, 1995[Abstract].

32.   Snedecor, GW, and Cochran WG. Statistical Methods (6th ed.). Ames, IA: Iowa State Press, 1967.

33.   Snodgrass, DR, Angus KW, and Gray EW. Rotavirus infection in lambs: pathogenesis and pathology. Arch Virol 55: 263-274, 1977[ISI][Medline].

34.   Starkey, WG, Collins J, Candy DCA, Spencer AJ, Osborne MP, and Stephen J. Transport of water and electrolytes by rotavirus-infected mouse intestine: a time course study. J Pediatr Gastroenterol Nutr 11: 254-260, 1990[ISI][Medline].

35.   Thouless, ME, DiGiacomo RF, Deeb BJ, and Howard H. Pathogenecity of rotavirus in rabbits. J Clin Microbiol 26: 943-947, 1988[ISI][Medline].

36.   Thouless, ME, Digiacomo RF, and Neuman DS. Isolation of two lapine rotaviruses: characterization of their subgroup, serotype and RNA electropherotypes. Arch Virol 89: 161-170, 1986[ISI][Medline].

37.   Touzani, K, Caüzac M, Vasseur M, and Alvarado F. Rheogenic Cl- conductance and Cl--Cl- exchange activities in guinea pig jejunal basolateral membrane vesicles. Am J Physiol Gastrointest Liver Physiol 266: G271-G281, 1994[Abstract/Free Full Text].

38.   Tzipori, S, Unicomb L, Bishop R, Montenaro J, and Vælioja LM. Studies on attenuation of rotavirus. A comparison in piglets between virulent virus and its attenuated derivative. Arch Virol 109: 197-205, 1989[ISI][Medline].

39.   Van Melle, G, and Robinson JWL Systematic approach to the analysis of intestinal transport kinetics. J Physiol (Paris) 77: 1011-1016, 1981[Medline].

40.   Vayro, S, and Silverman M. PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells. Am J Physiol Cell Physiol 276: C1053-C1060, 1999[Abstract/Free Full Text].

41.   Wright, EM. Genetic disorders of membrane transport. I. Glucose and galactose malabsorption. Am J Physiol Gastrointest Liver Physiol 275: G879-G882, 1998[Abstract/Free Full Text].

42.   Wyshak, G, and Detre K. Estimating the number of organisms in quantal assays. Appl Microbiol 23: 784-790, 1972[ISI][Medline].

43.   Zeng, CQ-Y, Wentz MJ, Cohen J, Estes MK, and Ramig RF. Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants. J Virol 70: 2736-2742, 1996[Abstract].


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