Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells.

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Omega. cm(2) between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.

The epithelial cell response to rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.

Replication as a determinant of the intestinal response to rotavirus.

Mucosal antibody induction by otherwise identical replication-competent and replication-incompetent rhesus monkey rotaviruses was compared. Although psoralen-inactivated virus induced diarrhea, the magnitude of the intestinal antibody response was severely attenuated compared with that of replicating rotavirus, as determined by assay of mucosal antibody-secreting cells. Rotavirus that was neutralized by monoclonal antibodies (anti-VP4 and -VP7) prior to inoculation was similarly ineffective at induction of specific antibodies in intestinal secretions. In contrast to genetically inactivated virus, antibody-neutralized virus did not induce diarrhea. In this murine model, viral replication is an important determinant of antibody induction. The diarrhea response is blocked by neutralizing antibodies, but the mechanism of action is not exclusively the inhibition of viral replication.

Rotavirus stimulates IL-8 secretion from cultured epithelial cells.

Rotavirus is the most important cause of severe gastroenteritis in children worldwide. We have investigated cytokine responses to rotavirus infection of cultured intestinal epithelial cells. Interleukin 8 (IL-8) is a chemotactic and cell-activating cytokine that is synthesized by epithelial cells and induced in response to bacterial enteric pathogens. Rotavirus inoculation increased IL-8 mRNA levels in cultured intestinal epithelial cells within 2 hr of infection. IL-8 secretion increased 10(2)- to 10(3)-fold by 8 hr postinfection. Secretion of TNF alpha or IL-1 beta, cytokines which themselves increase IL-8 secretion, was not induced by rotavirus, nor was that of TNF alpha, IFN alpha, IFN gamma, or IL-6. Neutralizing antibodies to TNF alpha or IL-1 alpha/beta did not affect the IL-8 response. Secretion of IL-8 was dependent on an intact viral capsid, as single-shell particles were inert. Neutralizing monoclonal antibodies (vp7-specific) that do not block cell attachment did block rotavirus stimulation of IL-8 secretion, indicating that attachment to the cell surface is not a sufficient stimulus to induce IL-8. Genetically inactivated rotavirus was also effective for IL-8 induction, indicating that viral replication was not required. These data suggest that epithelial cytokine IL-8 may be an important mediator of the host response to viral gastroenteritis pathogens such as rotavirus.

Rotavirus diarrhea is caused by nonreplicating viral particles.

Rotaviruses infect the villous epithelium of the small intestine and cause severe diarrhea in young children. The mechanism by which rotavirus causes diarrhea has not been elucidated. It has been hypothesized that rotavirus replication in the intestinal epithelium causes a loss of viable absorptive cells, leading to an imbalance of intestinal secretion and absorption. Cell destruction has generally been thought to result from rotavirus transcription and replication. However, the widely used heterologous murine model of rotavirus infection demonstrates minimal viral replication and histological changes limited to epithelial vacuolation on the distal villus despite the simultaneous occurrence of voluminous liquid diarrhea. We have genetically inactivated rotaviruses to test the importance of viral replication in the pathogenesis of rotavirus-induced diarrhea. We present direct evidence that transcription- and replication-defective rotaviruses cause diarrhea in an animal model. These findings suggest that rotavirus attachment or entry into cells is sufficient for the induction of diarrhea. The mechanism of rotavirus-induced diarrhea is therefore consistent with a viral toxin-like effect exerted during virus-cell contact.