New discovery on Norovirus – can form small, medium and large structures depending on the viral strain

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Stomach bugs come in different sizes.

Norovirus, best known for sweeping through daycares and cruise ships, can form small, medium, and large structures depending on the viral strain.

The discovery, reported June 10, 2019, in the journal Proceedings of the National Academy of Sciences, overturns nearly two decades of conventional wisdom about norovirus. Until now, the only structural data about the virus that scientists had came from a single, not particularly prevalent, strain.

“Everyone thought that all the strains would look about the same – like the one that was solved 20 years ago,” says Howard Hughes Medical Institute Investigator Leemor Joshua-Tor. “It turns out that they don’t!”

Joshua-Tor’s team used a microscopy technique called cryo-electron microscopy (cryo-EM) to visualize the shells of four viral strains, including one responsible for up to roughly 80 percent of norovirus outbreaks.

That strain was 71 percent larger (by volume) than the one previously reported.

Its shell was also decorated with a different pattern of molecular spikes.

Those structural details will be crucial for scientists working on vaccines or antiviral therapies to treat norovirus infection, says Joshua-Tor, a structural biologist at Cold Spring Harbor Laboratory (CSHL). Though norovirus causes about 21 million cases of foodborne illness in the United States every year, there are currently no approved therapies.

novovirus

At least one vaccine candidate is working its way through clinical trials now.

But CSHL study coauthor James Jung says scientists will need to take the virus’s newfound variation into account—so any new vaccine protects against a broad array of strains.

HHMI talked with Jung and Joshua-Tor to find out more about the “stomach flu,” the virus that causes it, and how they finally solved the structure of outbreak strains’ shells.

Q&A

What is norovirus?

Joshua-Tor: It’s a virus that causes gastroenteritis. It makes you throw up, causes diarrhea, and is extremely infectious.

Jung: It’s been reported that it takes as little 10 to 18 virus particles to start an infection.

Viral strains that cause “stomach flu” don’t all look alike
Different strains of norovirus can come in different sizes and molecular arrangements. An outbreak strain known as GII.4 (right) is larger than scientists expected. Credit: Joshua-Tor Lab

How many particles are in vomit?

Jung: I’m not sure, but it’s been estimated for a less-prevalent norovirus strain that a typical diarrheal sample contains approximately five billion viral particles per gram.

That’s a lot!

Joshua-Tor and Jung: Yes.

How does norovirus spread?

Joshua-Tor: It’s aerosolized when people vomit. Surfaces and food and water supplies can get contaminated.

That’s why it can spread so quickly among people in close proximity, like in daycare centers, nursing homes, and cruise ships.

What does norovirus look like?

Jung: It belongs to a family called Calciviridae.

The name comes from the Latin word “calyx,” which means cup-like cavity.

The surface of the virus has these spikes with depressions that look a bit like cups. So it’s basically a sphere with spikes sticking out.

And those spikes bind to human cells.

How many strains of norovirus exist?

Jung: 30 or more. A strain called GII.4 is responsible for the majority of all norovirus outbreaks.

But GII.4’s structure was completely unknown.

Why has no one focused on outbreak strains’ structure until now?

Jung: Before the resolution revolution of cryo-EM, the only way to solve high-res structures was through X-ray crystallography.

I tried crystallizing norovirus before we got our own cryo-EM microscope, but only one strain worked—it was the one that was previously solved. I think that’s why no one could solve the other structures.

Viral strains that cause “stomach flu” don’t all look alike
A microscopy technique called cryo-EM let scientists visualize the viral shells of four different strains of norovirus. Scale bar = 400 angstroms. Credit: Joshua-Tor Lab

How does cryo-EM work?

Joshua-Tor: You place your sample on a small copper grid and put it in this contraption that leaves a thin layer of the sample on the grid.

Then, you very quickly plunge it into liquid ethane, which freezes the sample and causes the water inside to become glass (we call it vitreous ice).

That means it doesn’t form crystals, which could interfere with the image. It also helps protect biological samples from radiation damage from the microscope’s electrons.

Next, you put the frozen grid into the microscope, which hits the sample with an electron beam. Then, you get images of your virus particles.

Cryo-EM has been around for a while. What’s new?

Joshua-Tor: In the last few years, cryo-EM has become very powerful—in terms of the speed of the cameras and the magnets that precisely control the electrons’ path.

When you hit a sample with electrons, it moves a little bit, so you can get a blurring effect. But [study coauthor] Nikolaus Grigorieff [of HHMI’s Janelia Research Campus] developed a technique that lets you collect movies instead of images and then computationally align the moving particles. All these things let us get much higher-resolution data than what we were getting before.

How did the norovirus outbreak strain differ from the known strain?

Jung: It turns out that the shell dimensions are different—a lot larger. It’s also made of a larger number of components.

Joshua-Tor: James calls it the shape-shifting virus. The organization of the spikes that talk to the cells in the body are positioned differently. You know how babies have toy balls with knobs on them to grip? That’s what the virus looks like, but the knobs all look a little different—so that’s important.

Why?

Joshua-Tor: We think how the knobs interact with human cells can affect virulence. And James discovered that there’s a zinc ion that helps the knobs organize in a particular way to promote binding to human cells.

Is it common for viral strains to differ in appearance?

Joshua-Tor: It’s not common, but it’s not unheard of. It wasn’t expected for norovirus.

How can knowing about these shells help us?

Jung: Current vaccine candidates have been made with the assumption that there’s only minor variation between strains.

Actually, the dimensions are completely different. Our findings could help people formulate a better vaccine.


How Norovirus Spreads

Norovirus is shed in the stool (feces) and vomit of infected people. Transmission usually results from the accidental ingestion of contaminated feces and vomit. This can occur in several ways:

Contact with surfaces contaminated with infected stool or vomit particles: 

The transmission of norovirus via inanimate objects such as shared medical equipment, computers and mobile devices puts the general public at risk.

, computers and mobile devices puts the general public at risk.

This is especially true in locations such as hospitals, nursing homes, schools, colleges and cruise ships.

Transmission through contaminated surfaces also increases the risk of infection for healthcare providers who care for infected patients, and for environmental service workers and custodians who clean and disinfect during an outbreak.

Contact with contaminated food and water: This can occur in restaurants and communal eating areas where an infected worker has contaminated food and water that is then ingested by customers.

Food may also be contaminated at any point, such as during its growing, processing or preparation.

Foods that are commonly responsible for outbreaks include leafy greens, fresh fruits and shellfish, particularly oysters.

The Role of Surfaces in the Spread of Norovirus

Hard surfaces play a particularly important role in the transmission of norovirus.

Studies have shown that the virus can persist in a dormant state on steel, wood, ceramic, plastic and glass for up to 28 days.

 The long persistence means that transmission may still occur long after a surface is contaminated.

Only a small amount of the highly contagious virus needs to be transmitted to cause an infection.

The rapid spread of norovirus was shown by the results of a study that assessed the ability of hands and cloths to spread norovirus-contaminated fecal material to other hand-contact surfaces, such as faucets, door handles and phones. 

The study showed that contaminated hands could transfer the virus to up to seven otherwise clean surfaces that hands often touch.

Another study used a harmless surrogate virus to mimic the spread of norovirus in an office building.

At the beginning of the day, the surrogate was placed on common surfaces such as doorknobs and tabletops.

After several hours, the hands of 40-60% of office workers had become contaminated. Many other commonly touched surfaces were also contaminated within a short period of time.

5. Preventing the Spread of Norovirus

Measures to prevent the spread of norovirus correspond to the ways the virus can be transmitted.

CDC guidelines issued in 2011 make five recommendations: hand hygiene, cleaning and disinfection of surfaces, thorough cooking of food, not preparing food when sick, and correctly washing soiled laundry. Two of the most important recommendations are cleaning and disinfection and hand hygiene.

Cleaning and Disinfection

Correct and thorough cleaning and disinfection of hard surfaces is critical to preventing the transmission of norovirus.

Surfaces should be regularly cleaned and disinfected with an EPA-registered disinfectant with approved claims against norovirus.

In general surfaces should be cleaned first to remove visible organic material such as contaminated feces and vomit, and then disinfected.

Once the disinfectant has been applied, it should be allowed to remain wet on the surface for the appropriate contact time.

The contact time, also known as the wet or dwell time, is the time the surface must stay visibly wet with the disinfecting solution in order for it to be effective against norovirus.

The product label will specify the contact time, which can range from 30 seconds to 10 minutes, depending on the disinfectant.

tes, depending on the disinfectant.

Some disinfectants containing quaternary ammonium chlorides as the antimicrobial ingredients may be effective against norovirus. Hydrogen peroxide-containing disinfectants are often more effective than quaternary ammonium chlorides.

However, the CDC commonly recommends a sodium hypochlorite (bleach) product, since bleach is considered the most effective disinfecting agent in norovirus outbreak situations.

The CDC recommends using a solution of 1,000–5,000 ppm household bleach, made by adding 5–25 tablespoons of household bleach (5.25%) to one gallon of water (equivalent to a 1:50 or 1:10 dilution) or any other disinfectant registered as effective against norovirus by the EPA. Users should always follow the product label’s directions for use if the product is registered against norovirus. 

Studies have documented the effectiveness of cleaning and disinfection to reduce norovirus load on surfaces and combat outbreaks.

In the study that demonstrated the spread of norovirus on surfaces, bleach was the only disinfectant that completely eliminated norovirus from surfaces. It did so only after the surface was first cleaned to remove organic soil.

The study also showed that use of a detergent that visibly cleaned surfaces did not inactivate norovirus but rather spread the virus on the surfaces.

In a hospital outbreak that affected 90 patients and 265 healthcare workers, a multi-strategy approach that included disinfecting with a 1:50 dilution of bleach was required to terminate the outbreak.9


More information: James Jung et al. High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations, Proceedings of the National Academy of Sciences (2019). DOI: 10.1073/pnas.1903562116

James Jung et al. High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations, (2019). DOI: 10.1101/580167

Journal information: Proceedings of the National Academy of Sciences
Provided by Howard Hughes Medical Institute

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