Institut National de la Santé et de la Recherche Médicale, Unité 510, Faculté de Pharmacie, Université de Paris XI, 92296 Châtenay-Malabry, France
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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 atIntestinal 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 · s1 · 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:
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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).
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RESULTS |
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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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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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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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DISCUSSION |
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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 ClPossible 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 |
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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.
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FOOTNOTES |
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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.
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