Role of Rotavirus NSP4 in childhood Diarrhoea

Author: Arturo Comba
Date: 27/03/2012


Arturo Comba
Diletta Giulia Giuntoli

Rotavirus is the most common cause of severe diarrhoea among infants and young children. By the age of five, nearly every child in the world has been infected with Rotavirus at least once and even though it’s an easily managed disease of childhood, nearly 500,000 infants die every year. However, with each infection, immunity develops, and subsequent infections are less severe; adults are rarely affected.
The virus is transmitted by the faecal-oral route. It infects and damages the cells that line the small intestine and causes gastroenteritis. Public health campaigns to combat Rotavirus focus on providing oral rehydration therapy for infected children and vaccination to prevent the disease.

Outline of the structure and viral replication

Rotavirus is a double-stranded RNA virus with a three-layered icosahedral protein capsid and no envelope, from the family of Reoviridae. There are 5 species of the virus (A, B, C, D, E) among which species A is the most common cause of infection in humans.
There are six viral proteins (VP1, VP2, VP3, VP4, VP6 and VP7) that form the virus particle (virion). In addition to the VPs, there are six nonstructural proteins (NSP1-NSP6), that are only produced in cells infected by Rotavirus. Among the NSPs, NSP4 is the main responsible for the pathogenesis of the disease: it is a viral enterotoxin that induces diarrhoea and it was the first viral enterotoxin to be discovered.

Rotaviruses replicate mainly in the gut, and infect enterocytes of the villi of the small intestine, leading to structural and functional changes of the epithelium. The triple protein coats make them resistant to the acidic pH of the stomach and the digestive enzymes in the gut.
The virus enters cells by receptor mediated endocytosis and forms a vesicle known as endosome. During the infection, Rotavirus produces mRNA for both protein biosynthesis and gene replication, then the progeny viruses are released from the cell by lysis.

Transmission and symptoms

Rotavirus is transmitted by the faecal-oral route, via contact with contaminated hands, surfaces and objects. Sanitary measures adequate for eliminating bacteria and parasites seem to be ineffective in control of Rotavirus, as the incidence of Rotavirus infection in countries with high and low health standards is similar.
The virus causes a mild to severe gastroenteritis. Incubation lasts two days, then symptoms start with low-grade fever and vomiting followed by 4 to 8 days of profuse watery diarrhoea.
Infection in newborn children, although common, is often associated with mild or asymptomatic disease; the most severe symptoms tend to occur in children six months to two years of age, the elderly, and those with compromised or absent immune system functions.
Death often occurs for dehydration.

Maldigestion and malabsorption of nutrients

Many studies demonstrate that Rotavirus induces a moderate net chloride secretion at the onset of diarrhoea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhoea (Vibrio Cholerae and Escherichia Coli).
One of the main effects of Rotavirus infection is a decrease in intestinal disaccharidase activities in vivo occurred with relatively intact intestinal brush border membrane (BBM).
Furthermore it has been noticed that the SGLT1-mediated Na+-D-glucose symport activity present in both villus and crypt cell BBM of intestine, although higher for villi than for crypt cells, was inhibited by Rotavirus in the absence of tissue damage. Despite this fact, experiments on rabbits demonstrate that glucose uptake value remains much higher for villi BBM in infected rabbits than for crypt cell BBM in control, non-infected rabbits, which would have been improbable if the enterocytes were crypt-like cells. Thus, the former hypothesis that Rotavirus infection kills off most of the mature enterocytes, leading crypt cells to invade the villus surface, generating a malabsorption type of diarrhoea, has been disproved.
Also, the inhibitions were unaccompanied by any effect on SGLT1 protein expression, which was again higher for villi than for crypt cells, strongly arguing against the crypt-cell invasion hypothesis. Because SGLT1 supports water reabsorption, the mechanism of Rotavirus diarrhoea may involve a generalized inhibition of Na+-solute symport systems, and hence of water reabsorption.
The NSP4 peptide has also been shown to instantaneously inhibit SGLT1, but not Na+-L-leucine symport activities. Hence, NSP4 is an effector directly causing glucose malabsorption during Rotavirus infection in vivo .
In conclusion, Rotavirus infection induces maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption, which can lead to diarrhoea.

NSP4 enterotoxin

It has been proved that NSP4 enterotoxin causes a moderate chloride secretion, but direct experimental evidence of this secretion is highly lacking. Rotavirus infects the mature enterocytes in the upper two-thirds of the villi of the small intestine, and the question arises – as with most luminal enterotoxins – as to the physical accessibility and binding capacity of the secreted NSP4 to the cells of the crypt region.
Thus, NSP4 indirectly stimulates chloride secretion. In neonatal mouse intestinal mucosal sheets, NSP4 was initially shown to potentiate cAMP-dependent Cl- secretion, but currents induced by NSP4 were small compared with those induced by cAMP, which led to the hypothesis of a cyclic-nucleotide-independent secretory diarrhoea.
The observation that addition of either NSP4 or carbachol (a cholinergic agonist that mobilizes Ca2+) to intestinal mucosal sheets induces transient, small and almost identical increases in Cl- secretory currents indicates that NSP4 determines a Ca2+-dependent Cl- secretory mechanism. Evidence has been gathered in favour of the mobilization of intracellular calcium associated with NSP4 expressed endogenously or added exogenously. Increasing intracellular calcium is known to induce a small, transient chloride secretion. This response contrasts with secretory diarrhoea in which activation of the cAMP- or cGMP-dependent intracellular second messenger pathways results in the sustained opening of the apical membrane channels leading to massive Cl- secretion.
It has also been proposed that NSP4-mediated Ca2+ mobilization may trigger the release of amines/peptides from intestinal endocrine cells as well as the release of cytokines, prostaglandins and nitrous oxide from the enterocytes. All these secreted compounds may, alone or together, activate the nervous system (ENS) in the intestinal wall, and hence stimulate intestinal chloride secretion. ENS involvement may explain how enterotoxins, which most likely do not reach the crypt region, can influence the secreting cells.
However, the possibility exists that NSP4 might exert positive as well as negative regulation of Cl- secretion, as did carbachol.
First, it has been found that after NSP4 pretreatment, addition of carbachol to intestinal mucosa had no additional effect on Cl- secretory currents. Such a result might be interpreted as a calcium-dependent Cl-secretion that makes cells refractory to re-stimulation by a second calcium-dependent agonist.
Second, NSP4 synthesized in Rotavirus-infected cells and NSP4 exogenously applied had been shown to increase intracellular Ca2+ through phospholipase C (PLC) activation. Such PLC activation can lead to transient chloride secretion through inositol (1,4,5) triphosphate (IP3) release. However, NSP4, like carbachol, could promote long-term inhibitory feedback through inositol tetrakisphosphate (IP4) production, preventing Cl- secretion from being sustained. Hence, the overall chloride secretory response will be determined by the imbalance between the stimulatory and inhibitory effects of NSP4 on the intestinal epithelium.
NSP4 has also been found to cause a disruption of tight junctions and a reduction in transepithelial electrical resistance accompanied by an increase in paracellular permeability to macromolecules of 20 kDa. However, the effect of increased paracellular permeability on fluid and electrolyte fluxes remains difficult to evaluate during Rotavirus diarrhoea in vivo.

Net chloride secretion

The few available data on intestinal Cl- secretion during Rotavirus diarrhoea have revealed discordance between in vitro and in vivo studies.
In vitro net Cl- fluxes, like net Na+ fluxes, in jejunal epithelium from piglets infected with human Rotavirus were secretory but did not significantly differ from those in non-infected animals.
Conversely, in vivo Cl- transport exhibited only net Cl- secretion in intestinal segments from Rotavirus-infected mice at 72 hours post infection, a time coinciding with both the increase in luminal Cl- concentrations and the peak of diarrhoeal severity.
Net Cl-secretion could not be due to reduced chloride absorption but rather to the presence of a secretory component. However, the increase in luminal Cl- concentrations is moderate, which would seem to be in line with observations that the ionic concentrations in the stools of Rotavirus-positive children are much lower than those found in the pure secretory diarrhoeas caused by secretagogues such as the enterotoxins of Vibrio cholerae and Escherichia coli.
While in these types of diarrhoeas it is widely accepted that loss of Cl- in the stools can be due to decreased absorption in the villus cells and/or increased secretion in the crypt cells, the mechanisms of Rotavirus-mediated diarrhoea appear to be rather different. Rotavirus infection fails to stimulate Cl- transport in crypt cell BBM, whereas it stimulates Cl- reabsorption in villus cell BBM.
As regards the overall chloride secretory response, these mechanisms appeared unable to explain the moderate increase in net Cl- secretion at the onset of Rotavirus diarrhoea. A good explanation would be that Rotavirus stimulates both Cl- influx and Cl- efflux in villi, since the chloride carrier is thought to function in both normal (absorption) and reversed (secretion) modes, depending on the direction of the chloride electrochemical gradient resulting from Rotavirus infection.
The presence of the Cl-/H+symporter may also explain why Rotavirus still results in diarrhoea in mice lacking the apical CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channel.

Mode of action of the viral and bacterial enterotoxins

The mechanisms by which Rotavirus and the NSP4 enterotoxin cause diarrhoea appear to be quite different from those described for bacterial enterotoxins, such as cholera toxin and the heat-labile and heat-stable enterotoxins of Escherichia coli, which cause "pure" secretory diarrhoea. The only common characteristic of bacterial and viral enterotoxins is that neither causes morphological damage. Bacterial enterotoxins are known to have no effect on coupled absorption of Na+ and glucose. On the other hand, Rotavirus has been shown to impair Na+-solute symport activities, hence contributing to massive water loss all along the small intestinal crypt-villus axis. NSP4 was also able to directly and specifically inhibit SGLT1. Unlike enterotoxigenic diarrhoea in which chloride malabsorption is coupled with chloride hypersecretion, leading to the massive loss of Cl-Rotavirus was found to cause substantial Cl- reabsorption in villi without stimulating Cl-transport in crypt. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of Rotavirus infection. NSP4 has no direct, specific effect on either intestinal absorption or secretion of chloride.
Finally, the intracellular mediators of chloride secretion were also different. In contrast to the sustained secretory responses induced by cyclic nucleotides, the Ca2+-dependent chloride secretory response induced by Rotavirus is transient and small, implying that negative signalling events may limit chloride secretion. The possibility that NSP4 may be able to exert both secretory and subsequent anti-secretory actions, as did carbachol, remains to be verified experimentally, but may explain the moderate loss of Cl-into the intestinal lumen at the onset of Rotavirus diarrhoea. All these considerations support the idea that NSP4 may act as an enterotoxin, but it would act as a viral enterotoxin that would function quite differently from bacterial enterotoxins by inducing mixed type rather than secretory diarrhoea.


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