Scientists make norovirus cultivation breakthrough

Baylor College of Medicine grew noroviruses in laboratory cultures of human intestinal epithelial cells.

It establishes a system in which strains can be grown, which will allow researchers to explore and develop procedures to prevent and treat infection and better understand norovirus biology.

Future significance ​

The work could lead to vaccines, therapeutics and measures to control the virus in humans. It is the first step in finding out how effective disinfectants and food processes are in inactivating infectious human norovirus.

Noroviruses do not grow in laboratory cultures that support growth of other viruses, such as transformed cells derived from cancerous tissues.

They are also species specific - human noroviruses only infect and cause disease in humans, and mouse noroviruses only do so in mice. Human noroviruses do not grow in mice or other small animal models used for research.

Norovirus is believed to be the most common cause of diarrhoea globally and the top cause of foodborne illness in the US.

NoroCORE, funded by USDA-NIFA and the National Institutes of Health, supported the work.

Role of bile​

The team grew norovirus in cultures that mimic the intestinal environment, where the virus naturally grows, by adding bile to cultures.

It is the first time that bile has been shown to be important for the replication of human intestinal viruses, they added.

Dr Mary Estes, Cullen endowed professor of human and molecular virology and microbiology at Baylor and emeritus founding director of the Texas Medical Center Digestive Diseases Center, said people had been trying to grow norovirus in the lab for a long time.

Estes, senior author of the study, said her idea was that they had not succeeded at growing noroviruses because they didn’t have the right cell type.

“We first showed that in patients with chronic norovirus infections, the virus could be detected in intestinal cells called enterocytes, but normal human enterocyte cells rapidly died when put into culture,” ​she said.

“A breakthrough came when we learned that Dr Hans Clevers’ team in the Netherlands had developed a method to make a new type of human intestinal epithelial cell culture system including enterocytes. ​

“These novel, multi-cellular human cultures, called enteroids, are made from adult intestinal stem cells from patient tissues. We anticipated that putting the virus in these non-transformed human cell cultures would let the virus grow.”​

Answering norovirus questions​

Methods used to study norovirus include RT-qPCR, electron microscopy, human volunteer studies and making inferences from viruses that can be cultured such as murine (mouse) norovirus.

It took them one year to get the human intestinal epithelial cultures growing well in the lab. Before they tested the cultures with noroviruses, they used another human gastrointestinal virus, rotavirus.

Estes said rotavirus grew well in the human intestinal epithelial cell cultures.

“Then, we tried the human norovirus and found that some strains would grow, but others wouldn’t. We suspected that still something was missing.”​

The researchers tried to improve growth of the viruses by adding to the cultures substances naturally present in the upper small intestine, the natural environment where the virus grows.

Dr David Y. Graham, professor of medicine and molecular virology and microbiology at Baylor and the Michael E. DeBakey Veterans Affairs Medical Center, said: “Viruses that cause gastroenteritis, such as rotavirus, utilize pancreatic enzymes to trigger their replication, but these enzymes had no effect on norovirus. ​

“We asked, if pancreatic enzymes were not important, was bile a key component allowing the virus to recognize where it was and replicate?”​

The human intestinal epithelial cultures are unique and the researchers anticipate they will allow them to answer questions, such as why one strain of norovirus infects one person, but not another.

Estes said the novel cultures retain properties of the different regions of the small intestine, and are physiologically active.

“Each culture is unique and reflects the genetics of the individual from whom the culture was established. This allowed us to show that some cultures, like some people, are susceptible to only one or to several human norovirus strains,” ​she said.

“These cultures will allow us to determine the mechanisms that restrict replication in some people, but not others. I predict this new culture system, changing certain conditions, will allow for the cultivation of other viruses or bacteria that we cannot grow at the moment. ​

“If we succeed, it will help us develop effective methods to prevent and treat infection, test vaccines, interrupt transmission, and better understand how these microbes infect people, respond to bodily defenses and evolve.”​

Enteroids are available with a Material Transfer Agreement from Baylor and the college has filed a related patent application.