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1 Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
2 Research Institute for Microbial Diseases, Osaka University, Japan
3 Laboratory of Physiology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Correspondence
Naoto Ito
naotoito{at}gifu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Center for Cancer Rersearch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Rotavirus infection in mammals is associated with blunting of the intestinal villi and mild infiltration of mononuclear cells in lamina propria (Greenberg et al., 1994). Rotavirus non-structural protein 4 (NSP4) and a synthetic peptide corresponding to aa 114–135 of NSP4 (NSP4114–135) have been shown to cause diarrhoea in suckling mice (Ball et al., 1996; Mori et al., 2002). NSP4 as a viral enterotoxin induces age- and Ca2+-dependent movement of halide ions across the plasma membrane in the initial cyclic nucleotide-independent pathway (Dong et al., 1997; Morris et al., 1999). Apart from the direct action of NSP4 on the mucosa at the onset of rotavirus diarrhoea, secondary mechanisms triggered by rotavirus replication and/or NSP4 are thought to sustain the diarrhoeal response. These mechanisms include mucosal destruction (Davidson & Barnes, 1979; Greenberg et al., 1994), villus ischaemia (Osborne et al., 1991), alteration of paracellular permeability and of expression of digestive enzymes (Jourdan et al., 1998; Dickman et al., 2000), and activation of the enteric nervous system (ENS) (Lundgren et al., 2000). The latter may explain how comparatively few infected cells at the villus tips can induce secretion of electrolytes and water from intestinal crypts. However, it is not known how rotavirus infection activates the ENS. Nevertheless, it is now recognized that the intestinal epithelium can release a wide range of biologically active compounds such as cytokines, prostaglandins and nitric oxide (NO) upon exposure to bacteria and viruses (Jung et al., 1995; Salzman et al., 1998; Rollo et al., 1999). Such mediators may influence intestinal fluid secretion either directly by acting on intestinal epithelial cells or indirectly through activation of the ENS.
NO is generated from L-arginine by a family of NO synthases (NOSs) (Nathan & Xie, 1994). There are three isoforms of NOS, named according to the cell type or to the condition under which they were first identified: endothelial NOS (eNOS), neuronal NOS (nNOS) and macrophage or inducible NOS (iNOS) (Nathan & Xie, 1994). The iNOS isoform, in contrast to eNOS and nNOS, is not expressed constitutively, but can be induced in a wide variety of cells by various stimulators such as bacterial lipopolysaccharide (LPS), gamma interferon and interleukin-1 (Nathan & Xie, 1994), leading to the generation of a large amount of NO. In the gastrointestinal tract, NO has been shown to regulate intestinal motility (Bult et al., 1990; Hoffman et al., 1997) and mucosal permeability (Kubes, 1992). However, accumulating evidence indicates that NO acts as a modulator of intestinal ion transport via either its activation of the ENS (Wilson et al., 1993; Izzo et al., 1998; Rhee et al., 2001) or its direct action on the intestinal epithelium (MacNaughton, 1993; Stack et al., 1996; Rolfe & Milla, 1999; Resta-Lenert & Barrett, 2002). However, direct involvement of NO in diarrhoea associated with intestinal pathogens, including rotavirus, remains to be shown.
A deeper understanding of how the host responds to rotavirus infection and to the actions of key viral proteins is important for elucidating the basis of viral pathogenicity. In the present study, we demonstrated that rotavirus infection can stimulate iNOS expression in the murine ileum. iNOS expression was also shown to be upregulated in the murine ileum and in macrophages upon exposure to NSP4. Furthermore, the enterotoxin domain of NSP4 was shown to be able to induce iNOS expression. These findings suggest that NO plays a role in rotavirus-induced diarrhoea.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To prepare inactivated rotavirus, strain SA-11 was mixed with 4'-aminomethyl-4,5',8-trimethyl psoralen (40 µg ml–1) and incubated on ice under UV light for 50 min (Groene & Shaw, 1992). The psoralen-inactivated SA-11 virus (PI-SA11) was characterized by failure to infect MA104 monolayers as determined by a focus assay 24–96 h post-inoculation (p.i.) and blind passages.
Rotavirus inoculations and diarrhoea score.
Five-day-old CD-1 mice were inoculated orally with 107 focus-forming units (f.f.u.) of simian SA-11, murine EW, avian PO-13 or PI-SA11. Mock inoculations were performed using EMEM without additives. Mice were monitored every day for 4 days. Diarrhoea was noted and a four-point qualitative scale was applied as follows: 1, normal brown formed stool; 2, yellow pasty stool; 3, yellow mixed liquid and solid stool; and 4, entirely liquid stool. A score of 2 or above was considered to be diarrhoea (Mori et al., 2001). For mRNA analysis, 4 cm long ileal segments were collected from control and infected mice, rinsed with supplemented DMEM and snap-frozen in liquid nitrogen.
NSP4s and culture conditions.
Full-length glutathione S-transferase (GST)-fused NSP4 (GST–NSP4) from the avian PO-13 strain, four GST-fused truncated PO-13 NSP4s corresponding to aa 86–169 (GST–NSP486–169), aa 109–169 (GST–NSP4109–169), aa 86–135 (GST–NSP486–135) and aa 86–169 truncated at aa 112–133 (GST–NSP486–169
112–133), GST-fused VP8 (GST–VP8) from the avian PO-13 strain and GST were expressed in Escherichia coli as described previously (Mori et al., 2002). Briefly, overnight cultures of E. coli DH5
strain transformed with recombinant pGEX-2T were diluted in fresh Luria–Bertani medium and grown to an OD600 of 0.3–0.4. Gene expression was induced by the addition of IPTG (Roche Diagnostics) to 0.1 mM and cultures were incubated for a further 12 h. Bacteria were pelleted by centrifugation at 4000 g for 10 min at 4 °C and resuspended in a sonication buffer [1 M Tris/HCl (pH 8.1), 0.5 M EDTA, 5 M NaCl] supplemented with 5 mM DDT and 1 % Triton X-100. Cell suspensions were then sonicated on ice with five short bursts of 10 s followed by intervals of 10 s. Debris was removed by centrifugation at 18 000 g for 30 min at 4 °C. Clarified cell lysates were passed through a GST 4B column (Amersham Pharmacia Biotech) three times. The column was washed three times and bound proteins were eluted with glutathione elution buffer [10 mM reduced glutathione in 50 mM Tris/HCl (pH 8.0)]. Proteins were then concentrated by ultrafiltration using Ultrafree-MC filtration devices with a 10 kDa nominal molecular mass limit (Millipore). Protein concentrations were determined using a Bio-Rad protein assay. Expression and purity of the recombinant proteins were determined by SDS-PAGE followed by Coomassie brilliant blue staining and Western blot analyses (data not shown, Borgan et al., 2003). Purified proteins were shown to be free of detectable endotoxin as determined by a Limulus amoebocyte lysate kit (Sigma). Production of polyclonal guinea pig anti-PO-13 NSP4 serum has been described elsewhere (Borgan et al., 2003). Peritoneal macrophages or confluent RAW264.7 cells were exposed to culture medium containing tenfold serial dilutions of GST–NSP4 (from 100 to 0.1 pmol), GST–VP8 (from 200 to 0.2 pmol), GST (from 200 to 0.2 pmol) or truncated GST–NSP4s (from 200 pmol to 0.2 pmol). LPS from E. coli 0127 : B8 (Sigma) was used as a positive control. For an antibody-dependent inhibition experiment, GST–NSP4 was pre-treated with polyclonal anti-PO-13 NSP4 guinea pig serum or with normal guinea pig serum for 1 h at 37 °C prior to cell treatment.
mRNA analysis.
Total RNA was extracted from macrophages treated with GST–NSP4 for 4 h or from frozen ileal segments using ISOGEN reagent (Nippon Gene). Contaminant genomic DNA was removed by DNase I (Takara). First-strand cDNA was synthesized from total RNA (5 µg) using an anchored oligo(dT)23 primer (Sigma) and Ready-To-Go You-Prime First-Strand beads (Amersham Pharmacia Biotech). cDNA (1 µl) was used as template for PCR amplification of iNOS message with Ex-Taq DNA polymerase (TaKaRa) for 35 cycles of 95 °C for 30 s, 68 °C for 60 s and 72 °C for 60 s. The iNOS cDNA was amplified using the following primers: sense, 5'-AGTGGGCCGAAGGATGGGCCTGGAG-3', and anti-sense, 5'-GTCTCACAGGCTGCCCGGAAGGTTTG-3'. The 
-actin gene was used as an internal control. -Actin primers and PCR conditions were as described elsewhere (Perrin et al., 1996). PCR products were resolved on a 1.5 % agarose gel containing ethidium bromide and visualized under UV light. PCR products were analysed further by DNA sequencing. Relative amounts of PCR products were determined by densitometry using NIH IMAGE 1.62 software and the ratio of iNOS mRNA to -actin mRNA was calculated.
Western blot analysis.
Macrophages treated for 24 h with GST–NSP4 or truncated GST–NSP4s were lysed with 100 µl of lysis buffer [25 mM Tris/HCl (pH 7.4) containing 150 mM Nacl, 1 % NP-40, 1 mM DTT and 50 µg ml–1 each of aprotinin, PMSF and leupeptin]. Soluble proteins (25 µg per lane) were separated by SDS-PAGE on 10 % gels and transferred to Immobilon PVDF transfer membranes (Millipore) by electroblotting. Membranes were then blocked overnight at 4 °C using PBS containing 0.1 % Tween 20 and 5 % non-fat dried milk. iNOS was detected by polyclonal antibodies to murine iNOS (diluted 1 : 1000; Sigma). An internal control, -tubulin, was detected by monoclonal antibodies to murine -tubulin (diluted 1 : 4000; Sigma). Signals were detected using Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products) and a luminescence image analyser (LAS-1000; Fuji Film).
Measurement of NO concentration.
The concentrations of the oxidized product of NO, nitrite (NO2–), in the culture media were determined using Griess reagent (Green et al., 1982).
Ex vivo treatment of ileum by GST–NSP4.
Four-day-old CD-1 mice were sacrificed under diethyl ether anaesthesia and 4 cm long ileal segments were harvested, flushed with DMEM and maintained in DMEM containing 2 % FBS and 2 µg gentamicin ml–1. After incubation for 1 h at 37 °C, the ends of the ileal segments were tied off and 50 µl of GST–NSP4, GST–VP8 or GST was inoculated. Ileal segments were then incubated for a further 4 h, followed by total RNA extraction and RT-PCR analysis as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Mori et al., 2001). We first sought to determine whether iNOS induction also occurred during the course of rotavirus-induced diarrhoea. Five-day-old CD-1 mice were inoculated orally with 107 f.f.u. simian SA-11, murine EW or avian PO-13, and diarrhoea was monitored daily as described in Methods. At 42 h p.i., when the severity of diarrhoea was greatest in suckling mice (data not shown), ileal segments were excised from the mice and subjected to RT-PCR analysis. -Actin mRNA was used as an internal standard control for RNA comparisons. Marked induction of iNOS mRNA expression was found in the infected mice but not in the control mice (Fig. 1). Nucleotide sequence analysis of the PCR products confirmed the amplification of authentic mouse iNOS. Induction of ileal iNOS mRNA expression by different rotavirus strains indicated that upregulation of iNOS mRNA expression is a common host response to rotavirus diarrhoea.
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). Expression of iNOS mRNA was markedly upregulated before the onset of clinical diarrhoea and was maintained at an elevated level for almost the entire course of diarrhoea. iNOS mRNA expression was, however, downregulated when diarrhoea was still at its peak (42 and 48 h p.i.), reaching an undetectable level by 48 h p.i. (Fig. 2).
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). Others have also confirmed that rotaviruses maintain their antigenic integrity after UV/psoralen inactivation (McNeal et al., 1999; Londrigan et al., 2003; Holloway & Coulson, 2006).Virus inactivation was confirmed by an immunofluorescent assay and two blind passages in cultured cells (data not shown). Identical amounts of PI-SA11 and untreated SA-11, corresponding to 107 f.f.u., were administrated orally to 5-day-old mice. Diarrhoea was monitored daily in order to assess whether the inactivated virus had any residual infectivity. In contrast to live viruses, no diarrhoea was observed in mice inoculated with PI-SA11 up to 5 days p.i., confirming that PI-SA11 was replication incompetent. In order to assess ileal iNOS mRNA expression, ilea were collected at 18 and 24 h p.i. and analysed by RT-PCR. Interestingly, in contrast to live virus, PI-SA11 was unable to induce ileal iNOS mRNA expression (Fig. 3a), indicating that ileal iNOS mRNA expression is associated with rotavirus replication.
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; Borgan et al., 2003) and is known to have strong enterotoxin activity, comparable to that of mammalian rotavirus NSP4 (Mori et al., 2002). Ileal segments excised from 4-day-old mice were maintained in supplemented DMEM and treated with GST–NSP4 (1000 pmol), GST–VP8 (2000 pmol) or GST (2000 pmol). Ileal segments were then incubated at 37 °C for 4 h followed by total RNA extraction and RT-PCR analysis. Expression of ileal iNOS mRNA was markedly induced after GST–NSP4 treatment but not after GST–VP8 or GST treatment (Fig. 3b
). Pre-treatment of GST–NSP4 with polyclonal guinea pig anti-PO-13 NSP4 serum significantly reduced the expression of ileal iNOS mRNA (Fig. 3b, lane 5), indicating the specificity of GST–NSP4 inducibility. These results showed that rotavirus NSP4 directly induces the expression of ileal iNOS mRNA.
iNOS expression and NO production by macrophages in response to GST–NSP4 exposure
iNOS expression and NO production in response to rotavirus NSP4 were assessed in murine peritoneal macrophages and RAW264.7 cells. In general, iNOS can readily be induced in macrophages by a variety of stimulators such as bacterial LPS and cytokines (MacMicking et al., 1997). When peritoneal macrophages were exposed to tenfold serial dilutions of GST–NSP4 (from 100 to 1 pmol) for 4 h, expression of iNOS mRNA was induced in a dose-dependent manner (Fig. 4a, lanes 3–5). The expression of iNOS mRNAs was undetectable in macrophages treated with 0.1 pmol GST–NSP4 (Fig. 4a, lane 6). Exposure of peritoneal macrophages to 200 pmol GST or GST–VP8 did not induce expression of iNOS mRNA (Fig. 4a, lanes 7 and 8). These results were further confirmed by analysis of iNOS protein and NO production by Western blotting and Griess reagent, respectively (Fig. 4b). Similarly, GST–NSP4 (from 100 to 1 pmol) markedly induced expression of iNOS protein and NO production in RAW264.7 cells in a dose-dependent manner (Fig. 4b, lanes 3–5). No effect was observed in GST- or GST–VP8-treated cells (Fig. 4b, lanes 7 and 8). GST–NSP4s from other avian rotaviruses, turkey strains Ty-1 and Ty-3 and chicken strain Ch-1 were also able to induce iNOS expression and NO production in RAW264.7 cells, indicating the universality of the stimulatory effect of NSP4 on macrophages, at least among avian rotaviruses (data not shown). To confirm further the specificity of NSP4 inducibility, GST–NSP4 was treated with normal or polyclonal anti-PO-13 NSP4 guinea pig serum for 1 h at 37 °C prior to stimulation of RAW264.7 cells. The results showed that anti-NSP4 serum, but not normal serum, significantly reduced the GST–NSP4 inducibility of NO production and iNOS mRNA expression in RAW264.7 cells (Fisher's test, P<0.02) (Fig. 5).
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). Macrophages treated with GST only exhibited low levels of iNOS protein expression and NO production (Fig. 6b and c, lane 1). This may have been mainly due to a low level of constitutive iNOS expression, as suggested by some reports (Hoffman et al., 1997). GST–NSP486–169, GST–NSP4109–169 and GST–NSP486–135 have been shown to be biologically active and to be able to induce diarrhoea in suckling mice (Mori et al., 2002). GST–NSP486–169, GST–NSP4109–169 and GST–NSP486–135 induced expression of iNOS protein and NO production in RAW264.7 cells (Fig. 6b and c, lanes 3–5). Although induction with the truncated proteins was slightly weaker than with GST–NSP4, these results were significantly different by Fisher's direct test (P<0.01) from the results obtained by GST treatment (Fig. 6b and c, lane 1). These proteins overlapped each other over aa 109–135. In contrast, no induction was observed in RAW264.7 cells treated with GST–NSP486–169112–133, in which aa 112–133 were deleted from GST–NSP486–169 (Fig. 6b and c, lane 6), indicating that the region aa 109–135 of PO-13 NSP4 has inductive activity for iNOS in macrophages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It is known that rotavirus infection can selectively induce chemokines and cytokines in intestinal epithelial cells (Rollo et al., 1999). We showed that expression of ileal iNOS mRNA is upregulated during diarrhoea caused in suckling mice by avian, simian and murine rotaviruses. The time course of ileal iNOS induction showed that expression of iNOS mRNA was rapidly upregulated within 6 h p.i. and remained elevated up to 36 h p.i. Although iNOS was induced before the onset of clinical diarrhoea, iNOS elevation paralleled the occurrence of diarrhoea. Decline of iNOS mRNA expression while diarrhoea peaked may call into question the potential role of NO in rotavirus diarrhoea. However, expression of iNOS is known to be regulated primarily by the de novo synthesis and stability of iNOS mRNA and protein (MacMicking et al., 1997; Rodriguez-Pascual et al., 2000; Carpenter et al., 2001). Furthermore, the diarrhoeal response seen after the decline of iNOS mRNA expression may be also explained by the changes in intestinal homeostasis and morphology caused by virus replication (Boshuizen et al., 2003).
Peaks of ileal iNOS mRNA expression were observed at 12 and 36 h p.i. The occurrence of two peaks during rotavirus infection is in agreement with results of a recent study showing two peaks of rotavirus replication in small-intestinal tissue at 1 and 4 days p.i. (Boshuizen et al., 2003). The differences between peaks observed in our study (at 12 and 36 h p.i) and those observed in a previous study (1 and 4 days p.i.) may be due to differences in the parameters measured (iNOS vs virus replication), as well as to mouse strains, rotavirus strains and the initial rotavirus load used to induce diarrhoea (107 f.f.u. vs 2x104 f.f.u.). Our results also suggest the dependency of iNOS mRNA expression on rotavirus replication, as shown by the inability of PI-SA11 to induce iNOS expression (Fig. 3a). This suggests that the accumulation of viral products is important for induction of ileal iNOS expression. As NSP4 only induced iNOS expression in the murine ileum, it is thought that NSP4 is at least one among other effectors (host and/or viral) that directly induce iNOS expression during rotavirus infection in vivo. In this respect, it is now known that dsRNA can also induce NO through a toll-like receptor 3-dependent pathway (Alexopoulou et al., 2001). Therefore, the possibility of roles of rotavirus dsRNA, other viral proteins and rotavirus-induced cytokines in the induction of ileal iNOS mRNA should not be excluded. Unfortunately, the lack of a reverse genetics system for rotavirus and the current impracticability of some available techniques such as RNA interference largely preclude the assessment of the potential roles of individual viral proteins in many steps of the pathogenicity of rotavirus.
The results of this study showed that NSP4 strongly and specifically induces iNOS expression and NO production in murine peritoneal macrophages and RAW264.7 cells, as well as in the murine ileum (Fig. 4). As a major part of innate immunity, iNOS is induced in macrophages in numerous diseases caused by various pathogens (MacMicking et al., 1997). Most bacteria induce iNOS expression in macrophages after invasion (Bekker et al., 2001) or through an LPS-mediated pathway (Xie et al., 1992). In contrast to the action of LPS on macrophages, NSP4 was able to induce significant iNOS expression, even at a concentration as low as 1 pmol, suggesting either that NSP4 is a potent stimulator for macrophages or that simultaneous NSP4-mediated events potentiate NSP4 action. It has recently been reported that intracellular calcium may contribute to iNOS induction in murine macrophages (Chen et al., 1998). Therefore, the potential role of NSP4-elicited intracellular calcium elevation in iNOS expression remains to be determined. On the other hand, the ability of NSP4 at a minute concentration (as low as 1 pmol) to induce iNOS expression in macrophages suggests that high-affinity NSP4 receptors are present on macrophages, although this remains to be proven.
NSP4 was also able to induce iNOS mRNA expression within 4 h in the murine ileum when the ileum was treated with NSP4 ex vivo. As the whole ileum was used to assess iNOS induction by NSP4, it is not clear whether the source of iNOS was intestinal epithelial cells or resident monocytes/macrophages. In fact, iNOS has been induced under various conditions in many intestinal cell lines such as the human adenocarcinoma cell lines HT-29/cl.19A and Caco-2 (Salzman et al., 1998; Resta-Lenert & Barrett, 2002) and in T84 colon epithelial cells (Korhonen et al., 2001). We tried to induce iNOS expression by NSP4 in Caco-2 cells, but no stimulation was observed (data not shown). However, the possibility of the role of intestinal epithelial cells cannot be excluded, as some in vitro models may not reconstitute all of the biochemical and molecular responses of the native epithelium. Furthermore, important species differences between the promoter regions of human and murine inos have been reported to be of functional significance (Nathan & Xie, 1994; Vallance & Charles, 1998). Whereas murine iNOS expression is rapidly induced upon exposure to certain stimulators, it has often proved difficult to induce functionally active iNOS in human cells and tissues in vitro (Vallance & Charles, 1998).
Many studies have demonstrated that rotavirus NSP4s from various animal and avian species have enterotoxigenic activities in suckling mice (Ball et al., 1996; Morris et al., 1999; Mori et al., 2002). Enterotoxigenic activity of NSP4 has been attributed to aa 109–135 (Ball et al., 1996; Mori et al., 2002). Our results showed that the inductive domain of iNOS on NSP4 exists within the enterotoxin domain, as demonstrated by experiments using a series of truncated NSP4s (Fig. 6). This suggests that NSP4 may exert its toxic activity by inducing NO production. Indeed, the enterotoxin domain of NSP4 is an active region with multifunctional activities such as intracellular Ca2+ mobilization, lipid membrane binding and Na+–D-glucose symporter inhibition (Dong et al., 1997; Halaihel et al., 2000; Huang et al., 2001). It is notable, however, that truncated NSP4s are less active than the full protein in inducing NO production, as larger amounts of truncated NSP4s were necessary for induction compared with the complete NSP4 (Fig. 6c). This finding has also been reported for NSP4 in inducing diarrhoea (Ball et al., 1996; Mori et al., 2002), suggesting that NSP4 needs to fold into its correct conformation for full functional activity.
Rodriguez-Diaz et al. (2006) have also investigated the role of NO during clinical and experimental rotavirus infection. They showed that iNOS mRNA is upregulated in the ileum upon rotavirus infection in mice. They also reported that NSP4 can inactivate Ca2+-dependent constitutive NOS, resulting in NO production within minutes in HT-29 cells. This observation is interesting, as we were not able to induce NO production in Caco-2 cells. This discrepancy might be explained in part by the fact that different cell lines respond to rotavirus and/or NSP4 in different ways. Holloway & Coulson (2006) have recently shown that rotavirus infection can activate p38 in Caco-2 and MA104 cells but not in HT-29 cells, suggesting that rotavirus activates distinct pathways in different cell lines. Moreover, Rodriguez-Diaz et al. (2006) did not show whether induction of Ca2+-dependent constitutive NOS was universal among different cell lines.
The association of iNOS induction with diarrhoea has been suggested for other infectious diseases such as Shigella and Cholera infections (Turvill et al., 1999; Rhee et al., 2001; Fasano, 2002). Elevation of iNOS mRNA before the onset of clinical diarrhoea suggests a role for NO in initiation of the diarrhoeal response. NO may later only sustain the diarrhoeal response, as suggested by the decline of iNOS mRNA expression while diarrhoea peaked. Indeed, three observations suggest that NO acts as a potentiator or initiator of rotavirus-induced diarrhoea. First, we found in this study that iNOS expression is elevated in response to reproductive rotavirus infection and that this elevation correlated with the occurrence of diarrhoea. Secondly, we previously showed that the enterotoxin NSP4, which is known to induce diarrhoea in neonatal mice within 2–4 h, was able to induce iNOS when it was administrated on its own to the murine ileum and that this induction occurred within 4 h. Thirdly, the fact that the enterotoxin domain of NSP4 (aa 109–135) is involved in NSP4 inducibility of iNOS may provide evidence that elevation of iNOS expression is associated with rotavirus diarrhoea. Future experimental approaches such as the use of L-arginine analogues, the use of specific iNOS inhibitors or the evaluation of rotavirus infectivity in iNOS-knockout mice are needed to address the role of NO in rotavirus pathogenicity.
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ACKNOWLEDGEMENTS |
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Received 4 October 2006;
accepted 6 March 2007.
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