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Rotavirus is a leading cause of diarrhea and vomiting, especially in children, which results in approximately 128,000 deaths per year. The virus triggers the disease by infecting enterocyte cells in the small intestine, but only a fraction of the sensitive cells have the virus. In the mid-1990s, scientists proposed that the small portion of infected cells promotes serious disease by sending signals that disrupt the normal function of nearby uninfected cells, but the nature of the signal remained a mystery.
In the current study published in the journal Science, a team led by researchers at Baylor College of Medicine found that rotavirus-infected cells release signaling molecules, identified as adenosine diphosphate (ADP), which binds its P2Y1 cell receptor to neighboring cells. Activation of P2Y1 by ADP produces signals called intercellular calcium waves in these uninfected cells. Disruption of ADP’s binding to its receptor reduced the severity of diarrhea in a mouse model of the disease, suggesting that P2Y1 targeting could be an effective strategy for controlling viral diarrhea in human populations.
“In our previous studies using fluorescent calcium sensors and time-lapse imaging, we found that rotavirus-infected cells display aberrant calcium signals that we can visualize as light pulses of intercellular calcium waves radiating from infected cells,” he said. corresponding author Dr. Joseph Hyser, assistant professor of virology and microbiology and member of the Alkek Center for Metagenomic and Microbiome Research at Baylor. “Calcium signaling was known to be associated with various aspects of rotavirus infection and our work revealed the dynamic nature of rotavirus-induced alterations.”
In the current study, the researchers conducted the experiments using a lower dose of the virus and noted that it was not only the virus-infected cells that exhibited dynamic calcium signaling, but the adjacent uninfected cells surrounding the infected cells also produced pulses. of football waves. which were coordinated with those of infected cells. This observation suggested that infected cells could trigger intercellular calcium waves in uninfected cells.
The researchers linked their observation to a concept proposed in the mid-1990s that suggests that rotavirus-infected cells send signals to nearby uninfected cells that disrupt their function, promoting diarrhea and vomiting.
“Our live fluorescent microscopy videos showing intercellular calcium wave signaling in rotavirus-infected and uninfected cells provided an unprecedented means of investigating the nature of the proposed signal, which had not been identified,” he said. first author, Dr. Alexandra L. Chang-Graham, an MD / Ph.D. student in the Medical Scientist Training Program who has completed her Ph.D. thesis working in the Hyser laboratory.
Find the signal
Chang-Graham, Hyser and their colleagues worked with three different laboratory models to identify the signal that triggers intercellular calcium waves on uninfected cells. They used a commonly used monkey kidney cell line to study rotavirus. They also worked with human intestinal enteroids, a cultivation system that summarizes many of the characteristics of human infection, and a neonatal mouse model of rotavirus infection and diarrhea. Their studies showed that suspected calcium wave triggers, such as prostaglandin E2 and nitric oxide, did not elicit a calcium response. Then they tested ATP and ADP, known calcium signaling mediators that had not previously been associated with rotavirus infection. They found that rotavirus-infected cells triggered intercellular calcium waves by releasing ADP that binds to its receptor, P2Y1, on neighboring uninfected cells. Elimination of the P2Y1 gene, which prevents ADP from signaling, reduced intercellular calcium waves.
“Through the three model systems we have consistently found evidence that rotavirus-infected cells signal cells not infected with ADP and that this contributes to the severity of the disease,” Chang-Graham said. “We consider a paradigm shift that the actual signal, ADP, wasn’t even on the radar before.”
ADP signaling is involved in triggering severe rotavirus symptoms
Further studies revealed previously unknown roles of ADP on rotavirus infection and replication, highlighting ADP as a major trigger for the multiple factors involved in severe diarrhea and vomiting caused by rotavirus. For example, the researchers found evidence that ADP signaling increases rotavirus infection, the expression of the inflammatory cytokine IL1-alpha, and the secretion of serotonin, an inducer of diarrhea. ADP signaling also increases the expression of enzymes that produce prostaglandins and nitric oxide, potentially causing the increases in those compounds seen in rotavirus infection. Preventing ADP signaling and intercellular calcium waves reduced the production of the aforementioned compounds.
“Finally, we determined that P2Y1 receptor inhibition reduces the severity of rotavirus-induced diarrhea in a mouse model,” Chang-Graham said. “By using intercellular calcium waves, rotavirus amplifies its ability to cause disease beyond the cells it directly infects. This is the first virus identified to activate ADP-mediated intercellular calcium waves. This may be a strategy other viruses also use to cause disease in their host. “
“Our findings add a new and very powerful signaling pathway into the causative mechanisms of rotavirus diarrhea,” Hyser said. “In terms of treatment, this is exciting because some drugs that target P2Y1 are currently undergoing preclinical testing such as anticlotting drugs. It is possible that such drugs could be reused, if proven to be safe for children, to be used for treat diarrhea caused by rotavirus infection. “
Other contributors to this work include Jacob L. Perry, Kristen A. Engevik, Francesca J. Scribano, J. Thomas Gebert, Heather A. Danhof, Joel C. Nelson, Joseph S. Kellen, Alicia C. Strtak, Narayan P. Sastri , Mary K. Estes and Robert A. Britton of Baylor College of Medicine. Melinda A. Engevik and James Versalovic are affiliated with Baylor and Texas Children’s Hospital.
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