Human noroviruses (HNoVs) are the primary cause of acute viral gastroenteritis worldwide and are responsible for up to 700 million cases and 200,000 deaths every year. However, despite this huge disease burden, there is no treatment or vaccine for the highly contagious infection, which is spread through vomit and feces. Making any therapeutic inroads has been hampered because scientists haven’t been able to identify exactly which type of cells in the gut epithelium act as the host for viral replication and spread, or shelter the virus during longer-term, chronic infection.
Studies in mice by researchers at Washington University School of Medicine (WUSM) have now thrown new light on norovirus infection, and shown that the virus specifically infects intestinal tuft cells, a rare chemosensory type of epithelial cell The findings highlight tuft cells as a potential target for the development of therapeutics or vaccines that could help to treat or prevent norovirus infection.
“Of the viruses worldwide for which there are no antiviral drugs or vaccines, norovirus arguably kills the most people,” comments Craig B. Wilen, M.D., Ph.D., an instructor in pathology and immunology at WUSM, who is first author of the researchers’ published paper in Science. “Norovirus is one of the deadliest human pathogens that we know the least about.…This study provides a therapeutic avenue to explore.” Studies by the WUSM team and collaborators are published in a paper entitled “Tropism for Tuft Cells Determines Immune Promotion of Norovirus Pathogenesis.”
Previous work by Dr. Wilen’s team had shown that a small population of epithelial cells acted as the reservoir for chronic MNoV infection, but it wasn’t clear how the infected cells differed from other epithelial cells. “… the reason for selective intestinal epithelial cell infection, how infected cells differed from adjacent cells in the intestinal epithelium, and why we seldom observed adjacent infected epithelial cells are unknown,’ the authors state. Further work by the team had then subsequently shown that norovirus infection requires the presence on host cells of a protein receptor known as CD300lf, but this receptor is primarily expressed on blood stem cells, rather than on gut epithelial cells. And while the researchers had determined that “rare isolated intestinal epithelial cells” could be infected by norovirus during chronic infection, expression of CD300lf on epithelial cells hadn’t previously been reported.
For their latest studies, the WUSM researchers used immunofluorescence to identify a scant population of CD300lf-expressing cells in the ilea and colons of mice, the shape of which suggested that the cells were tuft cells, a cell type found in hollow organs. Tuft cells are also known as brush, caveolated, multivesciular, or fibrillovesicular cells, and feature a characteristic long “tuft” of microvilli that protrudes into the intestinal lumen.
Further immunofluorecence analyses in mice infected with the MNoVCR6 strain of norovirus confirmed that the virus specifically infected the tuft cells in the animals’ intestines. No other epithelial cell types were infected. “Together, our immunofluorescence and flow cytometric analyses indicate that tuft cells are the physiologic target cell of MNoV in WT [wild-type] animals,” the researchers write. “This finding likely explains why we did not observe clusters of infected cells in the intestine, as tuft cells are isolated from one another, being surrounded by other intestinal epithelial cells.”
“We were most surprised that the virus infects such a rare cell type and that even with so few cells infected, the infections can be intense and easily transmitted,” comments Dr. Wilen. “In a single mouse, for example, maybe 100 cells will be infected, which is very few compared with other viruses such as the flu.”
Recent studies have found that tuft cells produce interleukin-25 (IL-25) and trigger type 2 immune reactions in response to infection by intestinal worm and parasite infections. Given this finding, the team reasoned that there could also be a relationship between type 2 immunity and norovirus infection. They found that pretreating mice with interleukin-4 (IL-4) or IL-25 rendered the animals more susceptible to a low dose of norovirus that would otherwise likely be unlikely to cause infection. IL-4 administration was also associated with significantly more virus particles being shed in the animals’ feces. “These results show that type 2 immune responses can enhance enteric viral transmission,” the team notes.
Previous work had in addition shown that establishment of a norovirus infection required an intact bacterial microbiome, and that broad-spectrum antibiotics that deplete intestinal bacteria could prevent norovirus transmission and persistent infection. However, the mechanism for this hasn't been understood. The WUSM team's RNA sequencing studies showed that antibiotic treatment resulted in reduced expression of tuft cell–specific genes in the colon, and reduced numbers of cells in the colon that expressed the tuft cell marker DCLK1. They also found that administering IL-4 and IL-25 induced tuft cell replication in the ilea of antibiotic-treated mice, but not in the colon. “These findings indicate that both type 2 cytokines and intestinal bacteria regulate tuft cells, albeit in a tissue-specific manner,” the researchers note.
The finding that intestinal bacteria contribute to tuft cell regulation led the team to query whether the antiviral effects of antibiotic therapy could be reversed by administering the type 2 cytokines. To test this, they first confirmed that pretreating mice with antibiotics for two weeks prior to administering a high dose of norovirus led to reduced rates of infection. They then showed that administering IL-4 or IL-25 to antibiotic-pretreated mice rendered the animals more susceptible to norovirus infection again. “IL-4 or IL-25 administration, prior to MNoVCR6 challenge, rescues viral infection in antibiotic-treated mice,” the team notes. “Both IL-4 and IL-25 significantly increased both the proportion of mice infected with virus and the magnitude of fecal shedding.”
The WUSM researchers claim their discoveries have important implications for understanding the pathogenesis of intestinal infections. In some cases, norovirus can trigger inflammatory bowel disease–like conditions, and the new findings pose the question of whether tuft cells might act to regulate inflammatory bowel disease–like phenotypes. The research results may also help to explain why people living in geographic regions where intestinal parasites and worms are more common are more likely to die of norovirus infection. It is also possible that other viruses infect tuft cells, the researchers add, “enabling these viruses to take advantage of type 2 immune responses to promote infection.”
Noroviruses can also persist in the intestine for months in healthy mice or human carriers, and the Science paper authors suggest that the tuft cells infected by the virus may somehow be escaping immune surveillance and providing a safe haven for the virus. “Our identification of MNoV tropism for tuft cells suggests that tuft cells represent an immune-privileged site for enteric viral infection in mice,” they write.
“This raises important questions about whether human norovirus infects tuft cells and whether people who have chronic norovirus infections and continue to shed the virus long after infection do so because the virus remains hidden in tuft cells,” Wilen said. “If that's the case, targeting tuft cells may be an important strategy to eradicate the virus.”
