Kinetics, Tissue Specificity and Pathological Changes in Murine Rotavirus Infection of Mice

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Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 ؋ 10 4 focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)-and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis.Rotaviruses are one of the most significant causes of gastroenteritis, malnutrition, and diarrhea in young children and animals (17, 28). Mortality rates are low in developed countries, where illness is usually self-limiting (64). However, each year more than 600,000 young children die in developing countries throughout the world (26). Rotavirus infection in children is mainly restricted to the small intestinal villus epithelium, resulting in the occurrence of total villus atrophy (3). Although rotavirus can infect older children and adults, diarrheal disease is primarily observed in children under 2 years of age (16,17,28).Rotavirus-induced diarrhea is thought to be caused by a combination of factors (55), which include a reduction in epithelial surface area, replacement of mature enterocytes by immature (crypt-like) cells (43), an osmotic effect resulting from ...

Section: Discussionsupporting

confidence: 73%

Section: Downloaded Frommentioning

confidence: 80%

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Changes in Small Intestinal Homeostasis, Morphology,and Gene Expression during Rotavirus Infection of InfantMice

Boshuizen

Reimerink

Male

et al. 2003

134

123

“…Villus ischemia. Although damage to the intestinal epithelium is minimal in rotavirus-infected mice, villus ischemia was observed in some studies (58,70). It was proposed that diarrhea could result from virus-induced release of an unknown vasoactive agent from infected epithelium, causing a local villus ischemia and subsequent functional damage to enterocytes (59).…”

Section: The Molecular Basis Of Diarrhea Inductionmentioning

confidence: 99%

Pathogenesis of Intestinal and Systemic Rotavirus Infection

Ramig

2004

314

230

Rotaviruses are responsible for significant gastrointestinal disease, primarily in children Ͻ5 years of age and the young of other mammalian species. Each year rotaviruses cause approximately 111 million episodes of gastroenteritis in children, which result in 25 million visits to clinics, 2 million hospitalizations, and 352,000 to 592,000 deaths. On a worldwide basis, nearly every child experiences rotavirus gastroenteritis by age 5, 1 in 5 visits a clinic, 1 in 65 is hospitalized, and 1 in 293 dies. Children in the poorest countries account for 82% of rotavirus deaths (60). This disease burden underscores a need for interventions such as vaccines. A vaccine was developed and approved, but recommendation for its use was withdrawn because of vaccination-associated adverse events (57). Additional information about the molecular biology, immunology, and pathogenesis of rotavirus infection will inform ongoing vaccine development efforts.Our understanding of rotavirus-induced diarrheal disease is incomplete compared to that of several other pathogens (e.g., cholera). Rotavirus diarrhea has been attributed to several different mechanisms, including malabsorption secondary to enterocyte destruction, a virus-encoded toxin, stimulation of the enteric nervous system (ENS), and villus ischemia (13,45). Over the past several years, numerous studies have addressed mechanisms of diarrhea induction at the cellular and tissue levels, and a new understanding of the mechanisms is beginning to emerge. Here I will briefly outline the new data and present our current understanding of how rotaviruses induce diarrhea in the infected host.Recent studies confirm sporadic case reports that rotavirus infection is not confined to the intestine as was generally assumed. Although systemic sequellae to rotavirus infection are apparently rare, reports have continually appeared in the literature, and recent work with animal models has begun to shed light on how the virus spreads to extraintestinal sites. Our current understanding of extraintestinal spread and infection will also be considered below. GENERAL CHARACTERISTICS OF ROTAVIRUS INFECTION AND DISEASE IN HUMANS AND ANIMALSRotavirus. Rotaviruses comprise a genus within the family Reoviridae. The rotavirion has a nonenveloped, complex, triple-layered capsid structure that surrounds a genome composed of 11 segments of double-stranded RNA. There are six structural proteins and six nonstructural proteins, each encoded in a unique genome segment except for nonstructural proteins 5 and 6 (NSP5 and NSP6), which are encoded in overlapping reading frames of a single segment. The rotavirus genus is divided into serological groups (A to E). Groups A to C infect humans, and all groups infect animals. All the information presented here is in regard to group A virus infections.Pathophysiology of rotavirus diarrhea. The enterocytes lining the small intestine are generally divided into two types: enterocytes and crypt cells (Fig. 1). Villus enterocytes are mature, nonproliferating cells covering the vill...

“…Rotavirus infects the mature enterocytes in the mid and upper villous epithelium of the small intestine, which ultimately leads to cell death, villous atrophy, and diarrhea. Mechanisms that have been proposed to explain the diarrhea include the following: malabsorption secondary to enterocyte death (9,16), villus ischemia (30,34), and a toxin-like effect of the nonstructural protein (NSP4) (2,11,38,40), and the enteric nervous system plays a key role in rotavirus fluid secretion (20).…”

mentioning

confidence: 99%

NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells

Tafazoli

Zeng

Estes

et al. 2001

112

The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20-to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.