June 1996
HOUSTON — A recently published study offers insight into the mechanism of rotavirus, possibly changing the direction of vaccine research.
Judith Ball, PhD, a research associate in the division of molecular virology at Baylor College of Medicine in Houston, and Mary Estes, PhD, professor, discovered here that a specific protein peptide caused rotavirus-like diarrhea in mice. The results were published in Science.
The protein, nonstructural glycoprotein number 4 (NSP4), is the first viral enterotoxin discovered, Ball told Infectious Diseases in Children.
"We are reporting that this protein by itself causes diarrhea," Ball said. "And not just the protein, but a specific piece of the protein, a peptide. We narrowed it to a very small portion. The disease caused by the toxin mimics rotavirus in that it is age- and dose-dependent."
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The investigators made the discovery while making an antiserum to NSP4. Within one to four hours after intraperitoneal or intraileal administration of NSP4, the mice developed diarrhea.
The researchers then tested a synthetic peptide corresponding to NSP4 residues 114 to 135. Younger mice showed signs of diarrhea within one to three hours, whereas older mice did not respond. The response increased when the peptide was delivered directly into the lumen of the intestine.
"These data ... reveal an age-dependent disease response to the NSP4 114-135 peptide that is similar to that seen in natural rotavirus infections or after inoculation of purified NSP4," the researchers wrote. "Regardless of the dose or the route of administration of the peptide, the kinetics of diarrhea induction were similar to those observed with purified NSP4. However, as compared with NSP4, the effective dose of NSP4 114-135 peptide was considerably higher. This may not be surprising, as the peptide may represent only a portion of the active domain or may not fold into the native conformation."
The protein mobilizes intracellular calcium, Ball explained. An increase in calcium causes increased chloride secretion, which in turn "causes secretory diarrhea," she said. "With chloride goes sodium, and with sodium goes water and you have diarrhea."
NSP4 acts as an enterotoxin, the Houston scientists concluded. This particular class of enterotoxin causes chloride secretion without damaging cells; a hallmark of rotavirus infection in mice is that it causes diarrhea without damaging the intestinal cells.
"On the basis of our results, we propose a model in which two intestinal receptors are required for symptomatic rotavirus infection," the researchers concluded. "One receptor binds rotavirus particles, resulting in virus entry and gene expression but not necessarily disease, whereas the second receptor is NSP4-specific. NSP4 expressed in infected cells would be released into the lumen and would interact with the second receptor on adjacent cells." This interaction would increase calcium levels, and thus, lead to diarrhea.
The concentration of NSP4 receptors declines with age, according to the study. Because the adult colon is able to handle the increase in fluid secretion, the virus may replicate in an adult and be excreted without causing disease.
The finding may have a significant impact on rotavirus vaccine research.
"We may have found one of the mechanisms of how rotavirus causes diarrhea," Ball said. "The antibodies are protective; when we give NSP4 antibodies to mice, it causes a significant reduction in disease. The current rotavirus vaccines [being tested] are 70% to 80% effective in reducing disease, but they do not reduce infection. By adding NSP4 to the vaccines, the reduction in disease could be even greater."
The impact of the findings may extend beyond rotavirus vaccines. This is the first viral enterotoxin discovered, suggesting that additional ones may be found that might enhance the understanding of other infections.
The next step is to find the receptor, Ball said; that study is currently in progress.
For more information, see:
- Ball JM, Tian P, Zeng CQ-Y, et al. Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein. Science. 1996;272:101-4.
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