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I spent last evening talking to friend, at an Amateur Radio Club, who told me he has recently recovered from the norovirus. I was demonstrating an oscilloscope to him, and he also handled the handbook. All this is now in the holdall I used to carry it to the Club. I took all the precautions I could, like washing my hands and not getting them near my lips, nose etc. So, my question is: how long should I wait before opening my holdall, to be sure any infection there is dead?
Well, for him the virus is already likely at minimum levels within his body. If he's recovered (pass the fever phase) then his immune system has already dealt with the majority of the viral bodies in his system, since the peak of the immune response comes after the peak of the viral infection density (ignore B):
How much of the virus was left in his system to actually spread is hard to guess. Sometimes infections never truly go away, but merely become undetectable by conventional testing. However, if we're to assume that there's at least some norovirus left to transfer to you then you should realize that the greatest chance for exposure has already come and gone.
The moment you were in the same room together, talking in close proximity, shaking hands (if you did), or generally being close is the most likely source of any infection you could have picked up. However, while most virii don't survive well outside of the body, the norovirus notably bucks that trend. The Hawaiian Department of Health (PDF) notes that the virus can definitely survive outside the body for several days on hard surfaces (though it will not grow), can survive being frozen, and that it only takes about 100 viral units to cause an infection (which could fit a few thousand times over on a grain of salt). Scientific American also notes that norovirus can survive for months or years in sources of drinking water.
What you've already done is pretty much what you should do to avoid infection. You've washed your hands, refraining from mucous membrane contact, etc.
Ultimately how long you should wait depends on where the norovirus currently resides. A week should be sufficient if all of the norovirus is contained on the book or on other surfaces that you're not making contact with. However, if it's already had contact with your skin or mucous membrane, then you're either already fighting it off or you're about to become ill. Unfortunately it's a lot easier to expose yourself than contain something you can't see, as the Mythbusters have done a good job of showing (Video).
In all likelihood if he was still infected enough to transmit the virus to other people by the time you two started handling the same equipment and you didn't use gloves, you've probably been exposed. However, exposure doesn't automatically mean you get sick. If you're not sick within a week afterwards, you've successfully fought it off. So, congratulations, perhaps!
Noroviruses are members of the Caliciviridae family. There are five genera in this family, one of which is Norovirus , and one species has been identified: the Norwalk virus ( ICTV, 2011 ). Noroviruses (NoV) have been genetically classified into five genogroups GII and GI are the genogroups primarily associated with human diseases, although NoV GIV has occasionally been implicated in outbreaks ( Zheng et al., 2006 ). Genogroup II.4 strains are responsible for most human NoV outbreaks, with up to 80% of infections being caused by this strain ( Widdowson et al., 2004 Leon and Moe, 2006 ).
Despite decades of research to identify a suitable in vitro cell culture system, there has been little success. Straub et al. (2007) described a three-dimensional in vitro system using human embryonic intestinal epithelial cell line INT-407 however, difficulty in adapting the system to other laboratories and the length of time required for the assay has precluded its widespread adoption. A method developed by the same group using CaCo-2 cells was reported recently ( Straub et al., 2010 ), but it has yet to be put into routine use. As a result, most of the information available on the structure of noroviruses has been obtained from studies conducted using recombinant norovirus-like particles that assemble spontaneously as recombinant VP1 and VP2 are expressed in insect cells ( Shoemaker et al., 2010 ). The advantages of working with this system are that the capsids do not contain RNA, and thus are non-infective, and that studies have shown that these particles are similar in size, shape, appearance and antigenicity to the infective virus particles ( Jiang et al., 1992 ).
Noroviruses are non-enveloped, single-stranded, positive sense RNA viruses. They are approximately 27-38 nm in diameter, with arch-like capsomeres on the capsids ( Prasad et al., 1994 ). The genome is 7.7 Kb, and contains three open reading frames: VP1 (the major capsid protein), VP2 (the minor capsid protein), and a non-structural polyprotein ( Jiang et al., 1993 ).
Norovirus pathogenesis: mechanisms of persistence and immune evasion in human populations
Noroviruses are important human pathogens known to cause epidemic outbreaks of severe gastroenteritis in communities, military barracks, cruise ships, hospitals, and assisted living communities, resulting in over 267,000,000 annual infections worldwide. Diversity within the norovirus genus allows this virus to persist in human populations, although a single genocluster, the GII.4 noroviruses, currently accounts for approximately 80% of all infections. Noroviruses bind to the polymorphic histoblood group antigens (HBGAs), which act as the putative cellular receptor, and strains from different genoclusters bind various HBGAs. Human challenge studies using viruses from different genoclusters have demonstrated that norovirus immunity is complicated and probably confounded by pre-existing exposure histories and variable immune responses. Evidence for both short-term and long-term immunity has been demonstrated, but the molecular mechanisms mediating differential immune responses in the face of infection remain unclear. Studies with virus-like particles from the GII.4 genocluster demonstrated that variation in and around the receptor-binding domain results in differential HBGA binding and altered antigenicity. These observations suggest that the norovirus capsid evolves to evade the memory immune response while retaining its ability to bind any of several HBGAs. In this review, we discuss how evolution within the capsid drives receptor switching and allows escape from herd immunity.
Norovirus is resistant to common disinfection practices, SEAS research finds
Experts said the norovirus can be susceptible to certain compounds like chlorine for disinfection but only to very high concentrations which are typically toxic and “corrosive” to surfaces.
Updated: May 11, 2021 at 6:12 p.m.
A professor from the School of Engineering and Applied Science discovered that a stomach virus can survive and continue to spread despite common sanitation practice.
The study, published last month with the National Institutes of Health, found that the norovirus – a highly contagious gastrointestinal virus – is resistant to disinfectant practices like ultraviolet light treatments and detergent solutions. Danmeng Shuai, an associate professor of civil engineering and the lead researcher of the study, said his results indicate that common decontamination procedures like using ultraviolet light to kill viruses in water-supply plants and cleaning surfaces with common household detergents may be ineffective.
The norovirus is considered the leading cause of foodborne illnesses, leading to anywhere from 19 to 21 million cases of vomiting and diarrhea illnesses in the United States each year, according to the Centers for Disease Control and Prevention. Shuai said the norovirus has more “protection” against outside stressors, like disinfectants and ultraviolet light, because it travels in vesicles that conceal it more so than other viruses that travel freely.
He said vesicles are like sacs outside of the cell that hide clusters of the norovirus inside them and make the virus more resistant to forms of disinfection like ultraviolet light. Ultraviolet light can be used to kill viruses in drinking water – a sanitation method that may really be ineffective following Shuai’s discovery of the protective vesicles.
“We need to revisit our current disinfection practices, because all the current guidelines are designed based on the disinfection of the free viruses,” Shuai said. “People didn’t know about the viral vesicles before so they never considered redesigning the current disinfection guidelines.”
Shuai said Mengyang Zhang, a doctoral student in SEAS, conducted experiments on norovirus particles by comparing how long single free and clustered virus particles survived against stressors like detergent. Shuai said Zhang and the other researchers used ultraviolet lights, detergents and freeze and thaw cycles to test the strength of the norovirus at a lab at the NIH.
Researchers shined ultraviolet light at varying strengths for different amounts of time on groups of virus clusters like the norovirus and free viruses to see which was more resistant, according to the study. Shuai said the virus clusters were more resistant to ultraviolet light, suggesting that ultraviolet disinfection may not be an effective method of killing the norovirus, which is commonly found in water supply plants that hold drinking water.
Shuai said they found that viral vesicles concealing the norovirus are “really, really robust,” and that the norovirus will survive even under extreme temperature changes since the vesicles did not decay in the freezing temperatures.
The researchers also found that the virus clusters in vesicles were 2.16 times more resistant to disinfection than the free viruses, according to the study.
He said this is just a “pioneer study,” and researchers still need to test other compounds like chlorine and ozone, which may be able to break down the virus.
“We need more study to understand that, but it brings up a possibility that these viral vesicles can be escaped from the current disinfection and sanitation processes and survive,” Shuai said.
Nihal Altan-Bonnet, a researcher at the NIH and the co-leader of the study, said when multiple variants of the norovirus travel together in a vesicle, one variant of the virus may not be able to make a specific enzyme, but the other variants can make it for the benefit of the entire vesicle.
Altan-Bonnet said the norovirus enters the cells in our bodies, replicates and then enters vesicles in the cell. She said the cell then sheds these vesicles which go and infect other cells.
“Inside each one of those vesicles is not one, but many norovirus particles, five, six norovirus follicles, maybe more,” Altan-Bonnet said. “And then those vesicles leave the cell, the cell remains behind intact, alive, keeping on shedding these vesicles. Then it’s these vesicles that go and infect another cell.”
Mengyang Zhang, a doctoral student at SEAS, said she was able to join the research team as a member of the GW/NIH Graduate Partnership Program.
Zhang, who joined the program in October 2019, said the work has helped her explore her interests in both biology and environmental engineering. She said environmental engineers often do not conduct these projects on virology because recognizing virus problems is not typically studied in their field.
“After I joined this program, I got very systematic training of virology at NIH,” Zhang said. “And also, I can utilize my environmental engineering background to do interdisciplinary research, so I can combine both of my backgrounds.”
Experts in biology and food sciences said the norovirus spreads in densely populated settings and has a protein code, making it more resistant to disinfectants than other viruses that are enveloped in a cell membrane, like the coronavirus.
Lee-Ann Jaykus, an assistant professor of food sciences at North Carolina State University, said the Environmental Protection Agency allows companies to label their disinfection products as anti-norovirus if they work against norovirus surrogates, viruses that are supposed to replicate human norovirus behavior, which can be misleading.
“So many of the products that are out there that services say ‘we have anti-norovirus activity’, well, they have anti-norovirus activity against the surrogates,” she said. “But if you were to test them on human norovirus, that likely would not translate.”
Christiane Wobus, an associate professor of microbiology at the University of Michigan, said alcohol-based hand sanitizers do not inactivate the norovirus. She said hand sanitizers are meant to dry out a virus cell’s membrane and break it open, but the norovirus’s protein code outside its membrane blocks this function.
“In case of SARS-CoV-2, you inactivate the virus by breaking the membrane because now the virus can get into the cell,” she said. “In the case of noroviruses, you break the membrane and free the particles, which then can still infect the host.”
Sejal Govindarao contributed reporting.
This post has been updated to correct the following:
The Hatchet incorrectly spelled Mengyang Zhang last name. It is now corrected. We regret this error.
The Norovirus: A Study in Puked Perfection
Today, The Guardian relayed one of those stunning medical stories that causes me to clean off my glasses and take another look to make sure I’m reading it clearly. They report that an outbreak of norovirus in Britain this winter has struck more than 1.1 million people with vomiting and diarrhea.
That’s right: 1.1 million. In Britain alone.
What is this fearsome bug, you may be asking, and why isn’t it the subject of a Hollywood horror movie?
Noroviruses are one of virology’s great open secrets. In a recent issue of The Journal of Infectious Diseases, Aron Hall of the Centers for Disease Control declared, “Noroviruses are perhaps the perfect human pathogen.”
Here’s what inspires awe in scientists like Hall.
Each norovirus carries just nine protein-coding genes (you have about 20,000). Even with that skimpy genetic toolkit, noroviruses can break the locks on our cells, slip in, and hack our own DNA to make new noroviruses. The details of this invasion are sketchy, alas, because scientists haven’t figured out a good way to rear noroviruses in human cells in their labs. It’s not even clear exactly which type of cell they invade once they reach the gut. Regardless of the type, they clearly know how to exploit their hosts. Noroviruses come roaring out of the infected cells in vast numbers. And then they come roaring out of the body. Within a day of infection, noroviruses have rewired our digestive system so that stuff comes flying out from both ends.
To trigger diarrhea, the viruses alter the intestinal lining, causing cells to dump out their fluids, which then gets washed out of the body–along with many, many, many noroviruses. Each gram of feces contains around five billion noroviruses. (Yes, billion.)
Noroviruses also make us puke. And if you can gather enough strength to think clearly about this, virus-driven vomit is a pretty remarkable manipulation of a host. Vomiting occurs when our nerves send signals that swiftly contract the muscles lining the stomach. Vomiting does us a lot of good when we’re hurling out some noxious substance that would do us harm. But repeated projectile vomiting of the sort that noroviruses cause serve another function: they let the viruses to find a new host.
To get us to throw up so violently, noroviruses must tap into our nervous systems, but it’s not clear how they do so. Here’s one particularly creepy hint: some studies indicate that during a norovirus infection, our stomachs slow down the passage of food into the intestines. In other words, they seem to load up the stomach in preparation for vomiting. Every particle of that stored food is a potential vehicle for noroviruses when it comes flying out of the mouth.
Once the norovirus emerges from its miserable host, it has to survive in the environment. Noroviruses have no trouble doing so, it seems. Fine droplets released from sick people can float through the air and settle on food, on countertops, in swimming pools. They can survive freezing and heating and cleaning with many chemical disinfectants. In 2010, scientists surveyed a hospital for noroviruses and found 21 different types sitting on a single countertop. It takes fewer than twenty noroviruses slipping into a person’s mouth to start a new infection.
This natural history makes for Olympic-level feats of transmission. In 2010, for example, nine members of a girl’s soccer team got sick with noroviruses while on a trip for a tournament in Oregon. The outbreak began with one girl coming down with stomach pains one Saturday evening. She moved from her hotel room to stay with a chaperone, where she then had diarrhea and vomited through the night. The chaperone took her back home in the morning (and also became sick later). Only on Tuesday did the rest of her team get sick.
Epidemiologists figured out that the first step in the transmission took place in the chaperone’s hotel room. There was a reusable grocery bag sitting in the bathroom–which the first girl never touched as she went in and out through the night. The next day, another chaperone got the bag and brought it to another hotel room for lunch. It contained sealed containers of chips, cookies, and grapes. Seven of the eleven people who ate that food got sick.
Another display of the norovirus’s tenacity came with a study of a New Zealand plane in which an infected passenger threw up on the floor of the economy section. A flight attendant cleaned up the mess, and over the next week, the plane continued to fly without any cases of vomiting. Nevertheless, the norovirus managed to infect new hosts. Out of 63 flight attendants who worked in the plane over the next six days, 29 got sick–an attack rate of 42.9%.
No one can say how the current outbreak in Britain got its start, but its timing is typical: January is peak norovirus season. Places where people are in close quarters are especially good incubators for the virus. The Queen Mary II, for example, is currently getting scrubbed down after a bad outbreak. But cruise ships are hardly the only place where noroviruses thrive. Schools get cleared out from time to time by the pathogens (the name norovirus comes from Norwalk, Ohio, where it was first isolated from a school during a 1968 outbreak). Nursing homes are fertile ground, too, in part because people there often have weak immune systems. While healthy people can clear out a norovirus after a couple of exhausting days, the virus can cling to people with weak immune systems for months or even years.
Noroviruses are so good at spreading that it’s quite likely that at some point in your life, you’ve had a norovirus infection. (You may have wrongly called it a stomach flu. Flu–short for influenza–only infects people’s airways.) It’s estimated that in the United States alone, noroviruses infect at least 23 million people a year. Seventy thousand of them end up hospitalized, and nearly 800 die. Things are worse in developing countries, where people are less likely to get rigged to an IV to get pumped full of fluids. It’s estimated that noroviruses kill 200,000 children under the age of five every year in developing countries.
It would be very nice if we only had to worry about getting noroviruses once and then could enjoy protection from them for the rest of our lives. Unfortunately, it seems that we only have a brief protection of perhaps a few months, and then we’re fair game again. As a strain of norovirus encounters this short-lived defense, it evolves new ways to evade our immune systems. A modified strain can then sweep around the world in as little as three months.
While some drugs show promise in blocking noroviruses from infecting cells, none have passed muster in a clinical trial on people. The best hope to put a real dent in the reign of noroviruses may be vaccines. Last year, scientists at Baylor College of Medicine reported that a vaccine could provide some protection against the virus. When people were exposed to noroviruses, 69% of people who got the placebo became sick, compared to only 37% of the vaccinated subjects.
A vaccine that leaves more than a third of people vulnerable to a virus is not exactly a silver bullet. But against such a perfect pathogen, even a little relief can ease a lot of pain.
For more information, check out norovirus expert Stephanie Karst talking about noroviruses with the gang at This Week in Virology. For viruses in general, see my book A Planet of Viruses.
(Update 5:30 pm: Changed England to Britain and fixed some typos. 7:30 am: corrected Norfolk to Norwalk, clarified timing of British outbreak)
Postscript, 1/5/13: When I wrote this post, I had no idea that, thanks to Reddit, it would draw the attention of hundreds of thousands of readers. A lot of those readers have asked how to get rid of norovirus. Looking over their questions, I realized that I should add some practical advice along with the natural history.
In my original post, I wrote that it resists bleaching. That’s a bit misleading, and it made a lot of readers worry that the virus was totally unstoppable. So I’ve revised the passage, changing “bleaching” to “chemical disinfectants.” Norovirus is one tough virus, and a little bleach may not be enough to wipe it out. You CAN kill noroviruses with a lot of bleach, although scientists can’t say for sure how much will work. The trouble comes back to what I mentioned above: they can’t raise human noroviruses in culture. So they do the next best thing and test out the noroviruses they can raise–ones that infect cat and mice. We can only hope that human noroviruses work the same way.
So–here’s what you can do to get rid of noroviruses, according to the Centers for Disease Control:
Misery-inducing Norovirus Can Survive for Months -- Perhaps Years -- in Drinking Water
Purple packages of pain: false colored (no, they're not purple in real life) transmission electron micrograph of human norovirus. CDC/Charles D. Humphrey. CDC Public Health Image Library ID 10708, click for link.
If there is a central circle of hell, I now know what's there: endless glasses of water spiked with norovirus that you must drink for eternity. Yet incredibly, some persons of Achilles-class bravery/stupidity actually signed up for this punishment of their own free will, and did so in the name of science. Brave souls, I salute you.
Because what these people helped discover is nothing short of spine-tingling: norovirus can survive at least 61 days in well water. Considering it takes only the number of virus particles that you can count on two hands to make you wish for death for about 24-48 hours, this is not good news. However, there is some good news, too, in the world of norovirus defense. More on that in a minute.
OK, so many of you are no doubt wondering: What the heck is norovirus?
A Pain in the Gut
Norovirus is Norwalk Virus, named for the Ohio town which in 1968 was home to the virus's first identified outbreak and which no doubt do not include this information in its Chamber of Commerce literature. Often called "stomach flu" or "24-hour flu", this awful malady has no relation to influenza virus, but has gained a reputation no less sinister in recent years. It is the agent responsible for innumerable cruise-ship "gastroenteritis" outbreaks and outbreaks at camps, state fairs, nursing homes, schools, and yes, even NBA locker rooms.
Anyone who's experienced it can tell you it's a bit like having all of your intestines' pain receptors activated at once, with uncontrollable nausea and/or diarrhea added as a special bonus. When I was in high school, every so often I'd experience twelve hours of intense pain along with nausea so powerful that I'd feel the urge to hurl even when nothing was left. This was followed by 12 hours of utter exhaustion. Then, I'd feel pretty much normal again and go right back to school, no doubt perpetuating the cycle since victims shed virus for several days after they recover. I'm pretty sure that it was norovirus.
I've never given birth, but if I ever do, it will be interesting to make the comparison. So far, the only other thing that's come close to the pain of norovirus infection is an unfortunate incident in which I was told that some people didn't need anaesthesia during their flexible sigmoidoscopy (aka colonoscopy lite) and I chose this option in a misguided money-saving move. Once they blew the air into my colon (I know, I know, TMI), it was like someone had flipped all the norovirus pain switches again. Needless to say, the next 5 minutes were among the longest of my life.
This virus is responsible for about nine out of 10 "stomach flu" cases in the U.S., and is probably responsible for about 50% of the cases of what people call "food poisoning". It takes fewer than 10 virus particles to make you sick, and the virus can be spread by sick people handling your food or water, or shaking your hand, or by you touching surfaces they've touched, or even by (I know, ewwww) aerosolization of their bodily fluids when they flush the toilet after a visit to the necessary room.
At one Boy Scout Jamboree in the Netherlands, scientists calculated each sick person infected 14 others before anything was done. After strict hygeine was imposed, each sick person infected a mere two others, which, the scientists soberly noted, was still not few enough to contain such an outbreak. In the NBA outbreak mentioned above, the CDC concluded there were at least two occasions on which norovirus was likely to have been transmitted to a new victim during a game.
A Simple Formula for Suffering
Let's back up a bit and look at what viruses are in general, so you can understand what noroviruses in particular actually are. Viruses are little packages of DNA or DNA's henchman RNA wrapped in a protein and/or fatty lipid coat. The protein coat, if it exists, is referred to as a "capsid", and individual virus particles are "virions". When present, lipid coats are more or less like our own cell membranes, and are often stolen from them by the virus.
Noroviruses are in the family Caliciviridae, whose members seem to specialize in making hits on terrestrial vertebrates -- everything from frogs on up. Another calicivirus -- Rabbit Hemorrhagic Disease Virus -- has been used for bio-control in Australia and New Zealand, while other viruses in the family -- like the beautiful hexagonal icosohedral Sapovirus, below, cause other forms of gastroenteritis in people. Norovirus has a more or less amorphous spherical capsid. You can see this in the photo at the top, where a few viruses that happen to have been sliced in half during the preparation for microscopy reveal the cross section of the virus.
The ghostly, graceful icosahedrons of Sapporo Virus, also called Sapovirus, in the Calicivirus family. Creative Commons GrahamColm. Click image for source and license.
"Calicivirus", which I so hope is pronounced "ka-leaky-virus" -- not unlike the titular greeting in this totally unrelated but awesome ditty -- name comes from calyx, which means a cup or goblet. The botanists in the room will recognize the term as the same one that refers collectively to the sepals of a flower, the sometimes, but not always, cup-shaped green leaves at the base of a flower. Some species apparently have a cup-shaped depression on their capsid surfaces.
Caliciviruses contain one single piece of single-stranded RNA in a protein capsid with no lipid envelope. Norovirus is the same, and its RNA encodes a mere two proteins, both used in making the capsid. It is utterly amazing to me that something so inconsequentially small and simple could cause such profound misery from such an efficient little package. If someone calculated a misery per base pair per person infected index, I think norovirus would be right at the top, considering Ebola virus clocks in at just under 19,000 RNA base pairs and might cause a few hundred cases a year at most (thank god), while norovirus contains a mere 7,500 but infects 21 million, hospitalizes 70,000 and kills more than 500 people in the U.S. alone every year. In developing countries, the virus kills about 200,000 children under age five annually. Not Cool, norovirus, Not Cool.
Unfortunately, norovirus also has a high mutation rate even by RNA-virus standards. The further bad news here is that having no fatty-lipid membrane means that the virus isn't killed very well by alcohol or detergents (which break down fats), though bleach and old-fashioned handwashing supposedly work well (Oh, old-fashioned handwashing, is there anything you can't do?). This is not good news for those that rely on alcohol-based hand sanitizers and wipes (something to think about next time you blithely swipe an alcohol-based wipe across the handle of your grocery cart or rub your hands with hand sanitizer). Obviously, this is one insidious virus.
Which brings us to the findings of two new studies.
Norovirus. Bar=50 nm. F.P. Williams, U.S. EPA
Scientists wondered how long well water -- from which about half the U.S. population draws its water -- would support noroviruses. The viruses could and have gotten into such water through leaking septic tanks or sewer lines, and in fact, when I was a reporter in Wyoming, I covered just such a case at a remote kids' camp. The results of this study were jaw-dropping. The scientists spiked water from an Atlanta well with a known quantity of the virus. Then they had (the sado-masochistic?) volunteers drink this water on day one, 4, 14, 21, 27, and 61. Volunteers were sickened by the water on each of these days, including day 61.
They didn't have enough money to subject the poor people to "testing" longer than that. But they did store and test the water for viral RNA contained in intact capsids up to 1,266 days later. That's nearly 3 1/2 years after spiking the water. There was no change in RNA levels over a year later, and only a small reduction after 3 1/2. That is one tenacious virus.
Since most ground and well water in the U.S. isn't treated prior to drinking, the scientists suggested we might want to start doing that.
In lieu of that (this country is home to a hatred of government regulation neatly encapsulated in New Hampshire's motto "Live Free or Die"), scientists are working on another approach: a vaccine. This is also important, as I've already mentioned, because a lot of people pick up the virus in other places, and seniors with weakened immune systems in long-term care facilities are particularly vulnerable.
As covered in Science late last year (original New England Journal of Medicine paper here), scientists have discovered that when one of the two viral proteins is produced by cultured cells, they spontaneously assemble (as they do in nature) into "virus-like particles" that contain no viral RNA payload and are thus non-infectious. But they look like norovirus from the outside (check out the photo in the Science article), and apparently look enough like it to our immune system that they can generate a partially-effective response.
Symptoms of norovirus infection appeared in just over two out of three of people exposed to both the virus and a placebo vaccine, but in only one in three of people given the real vaccine. Their symptoms were also less intense and took longer to develop. Well, it ain't perfect, but it's a good start. Porcelain-god worshipers everywhere will no doubt greet the news with the greatest relief.
The views expressed are those of the author(s) and are not necessarily those of Scientific American.
ABOUT THE AUTHOR(S)
Jennifer Frazer, an AAAS Science Journalism Award–winning science writer, authored The Artful Amoeba blog for Scientific American. She has degrees in biology, plant pathology and science writing.
An obstacle overcome
Scientists have been trying — and failing — to grow norovirus in the lab for more than 40 years. They had to make do studying similar viruses, or infecting nonhuman animals such as chimpanzees. Or they could recruit human volunteers to undergo norovirus infection for a study.
In August of 2016, researchers were thrilled to learn that a lab at Baylor College of Medicine in Houston, led by Mary Estes and part of the NoroCORE effort, managed to grow norovirus in human cells.
The keys to their success were using unmodified gut cells direct from human biopsies, and adding bile, a liver secretion that aids in digesting. Within three days of adding norovirus to the cultures, the scientists observed a 10,000-fold to 100,000-fold increase in the virus’s genetic material, indicating it was replicating itself. Now, Estes and her colleagues are studying how bile components make the cells vulnerable to infection.
Estes’s group is also training others to use their system, and scientists expect it will be useful both to study the basic biology of norovirus infection and to check how well medicines or disinfectants deactivate the virus’s ability to infect gut cells.
Noroviruses are the most common cause of gastroenteritis in the United States. Gastroenteritis, an inflammation of the stomach and the small and large intestines, may be experienced as nausea, vomiting, diarrhea, and stomach cramps. Although norovirus infection is sometimes referred to as “stomach flu,” gastroenteritis is distinct from flu, a respiratory illness caused by influenza virus. Gastroenteritis can be caused by bacteria or by a variety of viruses, including noroviruses. It is usually not fatal, but the illness can be serious for infants and the elderly.
Norovirus infection can by caused by eating contaminated food, such as uncooked fruits and vegetables or shellfish (especially oysters), drinking contaminated water, or direct contact with someone who is infected with norovirus. Outbreaks often occur in closed or institutional settings, such as child care facilities, schools, nursing homes, dormitories, and cruise ships, because the virus is very contagious, and it spreads rapidly through these environments. Noroviruses are found in the stool or vomit of infected people and can spread through food that has been contaminated by infected handlers.
Noroviruses are classified as members of a category of viruses known as the Calicivirus family. The caliciviruses consist of four groups, of which the noroviruses are the most important human pathogen. Caliciviruses are single-stranded, positive-sense RNA viruses. The caliciviruses have been difficult to study due to their inability to grow in a cell culture system and the lack of a good animal model system.
Norwalk virus is the best known member of the noroviruses, and it has made headlines as the cause of outbreaks of gastrointestinal illness on cruise ships. Following a bout of “winter vomiting” at an elementary school in Norwalk, Ohio in 1968, Norwalk virus was the first of the noroviruses to be identified as the causative agent behind an outbreak of gastroenteritis. Norwalk virus is a small virus that consists of a single strand of RNA, which comprises the genetic material of the virus, surrounded by multiple copies of a single protein assembled into a protective coat that is called the capsid.
CLINICAL DIAGNOSIS AND ENVIRONMENTAL DETECTION
Rapid identification of an outbreak is the key to effective infection control. While molecular methods offer a definitive way to establish etiology, these diagnostic tests may not be available in some clinical settings due to time delays or resource limitations. Kaplan's clinical and epidemiologic criteria ( Table 5 ), developed prior to the advent of molecular methods, can be used to rapidly identify norovirus outbreaks (168). The utility of Kaplan's criteria was reevaluated in 2006, and these criteria continued to prove useful in identifying norovirus outbreaks where molecular diagnostics were not easily accessible (169).
|1||Vomiting in more than half of symptomatic cases|
|2||Mean (or median) incubation period of 24 to 48 h|
|3||Mean (or median) duration of illness of 12 to 60 h|
|4||No bacterial pathogen isolated in stool culture|
Interpretation of norovirus diagnostic testing relies upon the quality of the specimens submitted for analysis and therefore requires that appropriate specimens are properly collected and handled (170). The optimal specimen for the diagnosis of norovirus infection is diarrheal stool. Specimens should be collected in a closed container within 48 to 72 h of the onset of symptoms, although norovirus may be detected in stool samples for 7 to 10 days or longer. Specimens should be refrigerated at 4ଌ prior to testing and frozen at ଌ or ଌ for long-term storage. Vomitus is an alternative specimen type that may be used to supplement stool sample testing during outbreak investigations. Collection and handling are the same as for stool specimens. Serum specimens are not recommended for routine diagnosis.
Norovirus Antigen Detection
A number of enzyme immunoassays (EIAs) are commercially available for the detection of norovirus GI and GII antigens in stool specimens. The most commonly performed EIAs are IDEIA Norovirus (Oxoid Ltd., Hampshire, United Kingdom) and Ridascreen Norovirus (R-Biopharm, Darmstadt, Germany). These solid-phase, sandwich-type immunoassays demonstrate a wide range of sensitivities and specificities ( Table 6 ). For example, the sensitivities and specificities for IDEIA Norovirus range from 38.0 to 78.9% and 85.0 to 100.0%, respectively. Similarly, the sensitivities and specificities for Ridascreen Norovirus range from 31.6 to 92.0% and 65.3 to 100.0%, respectively. Factors contributing to these differences in performance include the viral load present in the stool specimen and the viral genotypes represented in the sample set, as the assay antibodies show differential genotype affinities. These characteristics in turn may be affected by the clinical context of collection (outbreak versus sporadic cases), the timing of collection relative to symptom onset, and patient demographics (pediatric versus adult). Further sources of variability include the use of different EIA kit lots and assay iterations, known as generations (at least two generations for IDEIA and three for Ridascreen), and the use of different nucleic acid amplification tests (NAATs) as reference methods.
Performance of norovirus antigen enzyme immunoassays
|Study reference||Test||Study location(s)||Case context||Population(s)||Sensitivity (%)||Specificity (%)||Reference method (reference[s])|
|363||IDEIA a||UK||Outbreak||Not specified||55.5||98.3||Conventional RT-PCR (180, 364)|
|365||IDEIA||USA||Outbreak, sporadic||Pediatric, adult||39.0||100.0||Conventional RT-PCR d|
|193||Ridascreen c||Germany||Outbreak, sporadic||Pediatric, adult||34.6||65.3||Nested RT-PCR (366)|
|367||Ridascreen||Australia||Outbreak||Not specified||47.0||71.0||Conventional RT-PCR (368)|
|369||IDEIA||Australia||Outbreak||Not specified||66.0||85.0||Conventional RT-PCR (368)|
|370||IDEIA||Netherlands||Not specified||38.0||96.0||Conventional RT-PCR (189)|
|371||Ridascreen||Venezuela||Sporadic||Pediatric||60.0||97.5||Conventional RT-PCR (364)|
|372||IDEIA||Canada||Outbreak||Pediatric, adult||60.6||100.0||Composite e|
|172||IDEIA||European||Sporadic||Not specified||46.2||95.7||Conventional RT-PCR f|
|373||IDEIA||Spain||Sporadic||Pediatric||76.9||85.9||Conventional RT-PCR (189, 374)|
|171||IDEIA||USA, UK||Sporadic||Pediatric, adult||59.0||93.3||Composite g|
|375||IDEIA||Brazil||Sporadic||Pediatric||45.0||100.0||Real-time/conventional RT-PCR (184, 198)|
|213||IDEIA||Hungary||Sporadic||Pediatric, adult||78.9||100.0||Composite h|
|376||Ridascreen i||Brazil||Sporadic||Pediatric, adult||49.5||93.9||Conventional RT-PCR (377)|
|378||Ridascreen i||Brazil||Sporadic||Pediatric||92.0||83.3||Conventional RT-PCR (377)|
|379||Ridascreen i||Germany||Sporadic||Not specified||77.0||96.0||Composite j|
|380||Ridascreen i||Italy||Sporadic||Pediatric, adult||40.0||96.0||Real-time RT-PCR (116)|
Several studies that have evaluated the performance of EIA for detection of norovirus in outbreak investigations compared to sporadic gastroenteritis cases have shown that these assays are more sensitive in the outbreak setting, particularly if multiple samples are collected (171, 172). For example, in a large European multicenter study reported by Gray et al. (172), IDEIA had a 33.3% sensitivity and Ridascreen had a 44.4% sensitivity when two specimens per outbreak were tested. Sensitivity increased to 80.0% for both methods if ϧ specimens per outbreak were tested. Similarly, Costantini et al. (171) reported that IDEIA had a 44.1% sensitivity when three specimens per outbreak were tested and a 77.8% sensitivity when ϥ specimens per outbreak were tested. Based on statistical modeling, it has been estimated that at least six samples must be tested by EIA to achieve a 90% probability of detecting a norovirus outbreak (173). This minimum threshold of 6 outbreak samples has been adopted by the U.S. CDC and is recommended for outbreak management (174).
In 2011, the Ridascreen Norovirus third-generation test received Food and Drug Administration (FDA) approval for use in norovirus outbreak investigations. Notably, the intended use does not include the diagnosis of sporadic norovirus gastroenteritis cases. The package insert also acknowledges the relative insensitivity of EIA testing of limited sample numbers in outbreak settings. Compared to nucleic acid amplification tests, EIAs are generally simple to perform, do not require special molecular diagnostic laboratory facilities, and typically have a short turnaround time. For these reasons, the EIA is an attractive method for outbreak investigations, particularly in laboratories that lack molecular diagnostic capabilities. However, as nucleic acid amplification testing becomes commonplace in diagnostic and public health laboratories worldwide, real-time RT-PCR and other NAATs may replace the EIA entirely for outbreak investigation. This transition may be further hastened by the development of near-care and point-of-care molecular platforms capable of rapid sample-to-answer detection of norovirus RNA.
Rapid immunochromatographic assays.
Rapid norovirus antigen detection via lateral-flow immunochromatographic assays may provide an alternative to standard EIAs for stool screening in near-care or point-of-care settings. Several commercial rapid antigen assays are available for the rapid detection of GI and GII noroviruses, including Ridaquick Norovirus (R-Biopharm, Darmstadt, Germany) and SD Bioline Norovirus (Standard Diagnostics, Inc., Kyonggi-do, South Korea). Similar to the EIA literature, there is a wide range of reported sensitivities ( Table 7 ), and the assays and study designs are subject to the same sources of variability. Ridaquick sensitivities range from 17.0 to 83.0%, and SD Bioline sensitivities range from 23.0 to 92.0%. Both of these tests show high specificity: 87.5 to 100.0% for Ridaquick and 99.7 to 100.0% for SD Bioline. Given these performance characteristics, positive test results are reliable, although negative test results may require follow-up with a more sensitive NAAT.
Performance of norovirus rapid antigen immunochromatographic assays
|Study reference||Test||Study location||Case context||Population(s)||Sensitivity (%)||Specificity (%)||Reference method (reference[s])|
|382||Laboratory developed||Japan||Sporadic||Pediatric||69.8||93.7||Conventional RT-PCR (383)|
|384||Ridaquick a||Australia||Not specified||Not specified||82.0||100.0||Real-time RT-PCR (198)|
|385||Ridaquick||Netherlands||Not specified||Not specified||57.1||99.1||Real-time RT-PCR (195)|
|375||Ridaquick||Brazil||Sporadic||Pediatric||69.0||98.0||Real-time/conventional RT-PCR (284, 298)|
|386||Ridaquick||Australia||Sporadic, outbreak||Not specified||83.0||100.0||Composite b|
|387||Ridaquick||USA||Not specified||Not specified||61.4||100.0||Real-time RT-PCR (387)|
|379||Ridaquick||Germany||Sporadic||Not specified||69.0||97.0||Composite c|
|388||SD Bioline d||South Korea||Not specified||Pediatric, adult||90.2||100.0||Real-time RT-PCR e|
|389||SD Bioline||South Korea||Sporadic||Pediatric, adult||76.5||99.7||Real-time RT-PCR f|
|390||Ridaquick||Thailand||Sporadic||Not specified||48.2||87.5||Nested/real-time RT-PCR g|
|391||Ridaquick||France||Not specified||Not specified||17.0||100.0||Conventional RT-PCR (392)|
Molecular Diagnostic Tests
RT-PCR is the gold standard for the detection and typing of norovirus, and numerous conventional and real-time norovirus RT-PCR assays have been developed. The first-generation norovirus assays utilized a variety of primers based solely on the first described Norwalk virus genome and required RT in a separate tube prior to PCR (12, 175,). These assays underestimated norovirus genetic diversity and therefore did not perform well when applied to clinical specimens. The second-generation assays took advantage of sequences from additional norovirus strains and for the most part used primers directed at conserved regions of the viral polymerase (179,). Importantly, these assays required post-PCR analysis via hybridization probes or sequencing to improve sensitivity and specificity. The difficulty in designing broadly reactive primers to accommodate norovirus diversity was illustrated in a study comparing a set of five additional second-generation, conventional RT-PCR assays tested against a panel of stool specimens selected to cover a range of norovirus genogroups/genotypes (183). Although 84% of the specimens were detected by at least one assay, the sensitivity of individual assays ranged from just 52 to 73%. These conventional assays were optimized in a variety of different ways to improve detection (184,) however, there remained issues of assay complexity and postamplification specimen handling.
These limitations were addressed with the development of real-time RT-PCR for norovirus diagnostics. These assays used numerous detection methods, including SYBR green (190,), hydrolysis (TaqMan) (194,), and hybridization probes (203, 204). While many of these assays were directed at the viral polymerase gene, further sequence analysis revealed that a conserved region at the ORF1-ORF2 polymerase-capsid junction could also be used as an effective target for detection. Kageyama et al. described the first of these junction-targeting assays, which used two reactions to detect both GI and GII noroviruses (198). Similarly, Hohne and Schreier designed a two-reaction, real-time assay for GI and GII viruses using their own ORF1-ORF2 primer-probe sets (196). Importantly, this assay did not require a separate RT reaction.
To further minimize the reaction setup time and the potential for carryover contamination, GI and GII ORF1-ORF2 primer-probe sets were optimized for use in a single, multiplex TaqMan reaction (195, 197). In these assays, the probes were differentially fluorescently labeled to allow simultaneous detection and genogrouping. This multiplex, multiprobe approach directed at ORF1-ORF2 has also been used with GII/GIV TaqMan probes (199), GI/GII/GIII TaqMan probes (205), and GI/GII hybridization probes (204). It remains unclear, however, whether immediate norovirus genogrouping is important for outbreak control or clinical management. Simple, broadly reactive assays (177, 206) may be more important in the acute setting. Alternatively, the routine use of assays that detect and type norovirus may simultaneously allow laboratories to more rapidly monitor epidemiological patterns and highlight geographic regions or communities requiring further investigation (207).
Given the numerous real-time RT-PCR assays available for norovirus detection and genotyping, well-controlled, comparative studies similar to earlier work by Vinje et al. (183) are required to accurately determine the relative performance characteristics of these assays. Vainio and Myrmel (208) provided the first of these studies, by looking at two assays described above (192, 196) as well as two assays initially designed for screening of shellfish (209, 210). After a detailed analysis, the duplex, GI/GII, real-time TaqMan RT-PCR assay designed by Jothikumar et al. (209) was selected for in-house use. Compared to a conventional nested approach (208, 211), this assay, which employs modifications of the primer-probe sets reported by Kageyama et al. (198), had a clinical sensitivity of 91%. These results were superior to those of the SYBR green assay reported by Richards et al. (80%) and slightly inferior to those of the TaqMan assay reported by Hohne and Schreier (93%) but with the advantage of detection of GI and GII noroviruses in a single reaction. This study was performed on specimens from norovirus outbreaks in Norway, so additional work will be required to account for norovirus diversity in different populations. Furthermore, this assay was selected based on both clinical and practical considerations, yet it was the only assay evaluated that was capable of single-tube identification of multiple genogroups. Future work is required to provide a more comprehensive analysis of the numerous real-time norovirus assays now available with these characteristics.
Commercial RT-PCR assays.
Several commercial RT-PCR reagents are available for norovirus RNA detection ( Table 8 ), although comparisons with one another or the large number of laboratory-developed norovirus RT-PCRs are not widely available. The Argene Calicivirus/Astrovirus consensus test (bioMérieux, Marcy l'Etoile, France) is a second-generation RT-PCR assay that requires a detection step via microplate hybridization using biotinylated probes. This test can identify caliciviruses and astroviruses but cannot distinguish between noroviruses and sapoviruses (212). Kele et al. used selected samples from sporadic cases of gastroenteritis in Hungary to evaluate the performance of Argene Calicivirus/Astrovirus Consensus and SmartNorovirus (Cepheid, Sunnyvale, CA), a set of primers and differentially labeled probes that allow real-time PCR detection and differentiation of GI and GII noroviruses by real-time RT-PCR (213). When true positives were defined as at least one positive RT-PCR result, the sensitivities of Argene Consensus and SmartNorovirus were 92.8% and 91.2%, respectively.
Commercial norovirus RT-PCR assays
|Argene Calici/Astrovirus Consensus||bioMérieux, Marcy l'Etoile, France||RT-PCR ELOSA a|
|SmartNorovirus||Cepheid, Sunnyvale, CA||Real-time RT-PCR|
|RealStar Norovirus||Altona Diagnostics, Hamburg, Germany||Real-time RT-PCR|
|Ridagene Norovirus||R-Biopharm, Darmstadt, Germany||Real-time RT-PCR|
|AccuPower Norovirus||Bioneer, Daejeon, South Korea||Real-time RT-PCR|
|Norovirus real-time RT-PCR||Shanghai ZJ Bio-Tech, Shanghai, China||Real-time RT-PCR|
R-Biopharm (Darmstadt, Germany) offers two internally controlled, norovirus real-time PCR assays, Ridagene Norovirus, for qualitative detection of GI and GII noroviruses, and Ridagene Norovirus I&II, which both detects and differentiates GI and GII noroviruses in a single reaction. When the AccuPower Norovirus real-time PCR assay (Bioneer Co., Daejeon, South Korea), another internally controlled assay for detection of G1 and GII noroviruses, was compared to Ridagene Norovirus, there was 99.0% (96/97) positive agreement and 95.1% (175/184) negative agreement (214). Similarly, comparison of the Ridagene Norovirus I&II assay with conventional RT-PCR using stool specimens from distinct outbreaks in Victoria, Australia, in 2012 and 2013 revealed 98% sensitivity (85% [11/13] for GI and 100% [87/87] for GII) and 98% (98/100) specificity (215).
Future comparative studies with large, globally distributed sets of stool samples from sporadic gastroenteritis cases and outbreak settings will be required to better define the performance characteristics of these commercial reagents.
Multiplex PCR/RT-PCR tests for diarrheal pathogens.
The development and widespread use of commercial, highly multiplexed molecular diagnostic technologies have revolutionized testing for infectious diseases. Although initial efforts were dedicated to the design of respiratory virus panels, the focus has now shifted to gastrointestinal pathogens, with a number of manufacturers developing multiplex panels for diarrheal disease. The first of these panels is the xTAG Gastrointestinal Pathogen Panel (GPP) (Luminex, Austin, TX). In addition to norovirus genogroups I and II, this assay detects rotavirus A, adenovirus 40/41, Giardia, Cryptosporidium, Entamoeba histolytica, Campylobacter, C. difficile toxin A/B, Salmonella, Shigella, Vibrio cholerae, Escherichia coli O157:H7, as well as enterotoxigenic and Shiga-like toxin-producing E. coli. This method utilizes multiplex RT-PCR followed by target-specific primer extension for the addition of oligonucleotide hybridization tags. The tagged amplicons are then specifically hybridized to a set of microspheres with unique spectral signatures and coupled to capture sequences complementary to the tag sequence. Finally, the captured amplicons are labeled with a detection reagent, and bead-bound amplicons are counted via flow cytometry. This liquid-based array approach allows a significant level of multiplexing however, it is laborious, it has a long turnaround time, and, because several steps require the handling of amplicons, there is a high risk of contamination.
A number of studies utilizing the xTAG GPP have been carried out, and the performance characteristics of the norovirus component of the assay are summarized in Table 9 . Claas et al. demonstrated good sensitivity (GI, 100% [9/9] GII, 92.5% [62/67]) and specificity (GI, 100% [642/642] GII, 97.6% [570/584]) compared to real-time RT-PCR, although it is important to note that all xTAG GPP testing in this study was performed by the manufacturer (216). Wessels et al. also showed good sensitivity (94.4% [17/18]) and specificity (100% [375/375]) compared to real-time RT-PCR (217, 218). Studies by Mengelle et al. and Navidad et al. are difficult to interpret, as very few norovirus reference method-positive specimens (six total) were identified (219, 220). Finally, although Kahlau et al. had a large number of xTAG GPP norovirus-positive specimens, confirmatory RT-PCR testing was performed on only a subset of positive specimens, and none of the xTAG GPP-negative specimens were tested with the reference method (221).
Norovirus detection using multiplex gastrointestinal pathogen panels
|Study reference||Test||Study location(s)||Case context(s)||Population(s)||Genogroup||Sensitivity c (%)||Specificity (%)||Reference method(s) (reference)|
|216||xTAG GPP a||International||Sporadic||Pediatric, adult||I||100.0||100.0||Real-time RT-PCR (218)|
|219||xTAG GPP||France||Sporadic||Pediatric, adult||I or II||0.0||93.6||ImmunoCardSTAT! b|
|220||xTAG GPP||United States||Sporadic, outbreak||Pediatric, adult||I||NA||100.0||Real-time RT-PCR (202)|
|217||xTAG GPP||Netherlands||Sporadic||Not specified||I||NA||100.0||Real-time RT-PCR (218)|
|222||TaqMan Array||Tanzania, Bangladesh||Not specified||Pediatric||II||100.0||96.2||RT-PCR Luminex (223)|
|224||BioFire FilmArray GI Panel e||United States||Not specified||Not specified||I or II||96.2 d||99.8||xTAG GPP, f real-time RT-PCR (202)|
Another multiplexing approach involves the use of TaqMan Low Density arrays (Life Technologies, Grand Island, NY), microfluidic cards comprised of 384 wells divided into 8 zones of 48 wells that are preloaded with a panel of singleplex TaqMan assay mixtures. Ports on the array allow extracted nucleic acids from 8 samples to be delivered to each zone for testing. Liu et al. (222) developed a gastrointestinal TaqMan array that detects 19 enteropathogens and includes the norovirus GII primer-probe set reported by Kageyama et al. (198). Compared to a laboratory-developed RT-PCR Luminex assay that also utilizes the GII primer and probe sequences of Kageyama et al. (223), the TaqMan array demonstrated 100% (31/31) sensitivity and 96.2% (75/78) specificity for norovirus GII.
While the xTAG GPP and TaqMan array methods are of high complexity and require separate nucleic acid extraction prior to amplification and detection, the BioFire FilmArray (bioMérieux, Marcy l'Etoile, France) is a moderate-complexity, 60-min sample-to-answer system that performs sample preparation, amplification, and detection in a single disposable pouch. The FilmArray GI panel detects 22 bacterial, protozoan, and viral targets, including norovirus GI/GII, although like the xTAG GPP, the FilmArray assay does not distinguish between genogroups. Khare et al. evaluated the FilmArray GI panel and showed 96.2% (52/56) sensitivity and 99.8% (441/442) specificity for norovirus GI/GII compared to a composite reference that included the xTAG GPP (224).
Additional large independent studies will be required to further characterize the norovirus component of these panels as well as other multiplex gastrointestinal panels currently in development. An important preliminary finding in these initial studies is the identification of infections with multiple pathogens. The evaluation of the clinical consequences of these coinfections will be an important area of future investigation.
In addition to the wide variety of RT-PCR-based amplification assays, isothermal PCR alternatives have also been developed for norovirus detection. Several groups have designed nucleic acid sequence-based amplification (NASBA) strategies by using previously reported primer pairs modified for NASBA compatibility (225,). A small study by Houde et al. (226) determined that NASBA and RT-PCR using the GII primer sets described by Kageyama et al. (198) showed equivalent analytical sensitivities but that the NASBA assay provided less consistent signals. Many NASBA formats, including those described above, require an additional product detection step. However, Patterson et al. (229) designed a NASBA assay using a molecular beacon probe that allowed real-time detection. This assay was 88% sensitive compared to conventional RT-PCR, suggesting that the assay requires further optimization. A norovirus NASBA assay, Swiftgene Norovirus GI/GII, is commercially available in Japan (Kainos Laboratories, Tokyo, Japan).
The final, real-time, non-PCR, nucleic acid-based approach for norovirus diagnosis is RT–loop-mediated isothermal amplification (RT-LAMP) (230,). Fukuda et al. (230) described a two-reaction, GI- and GII-specific RT-LAMP assay also using primers directed at the ORF1-ORF2 junction. Compared to conventional RT-PCR (184), the RT-LAMP assay had 100% clinical sensitivity. Based on this work, a commercial assay, Loopamp Norovirus GI and GII, was developed (Eiken Chemical, Tokyo, Japan) (232). One advantage of this approach is that detection is performed via inexpensive, real-time turbidimetry or simple, endpoint, visual examination. Future work will be required to compare the performance characteristics of RT-LAMP to those of real-time RT-PCR.
How does Norovirus Spread?
Lydia Drumright from Imperial College London is undertaking a new study to find out how the virus infects people and how long they remain infectious for. Kat Arney spoke to her.
Kat - So, tell us a little bit about what do we know so far about kind of the life history of norovirus. How it infects us and where it goes in populations?
Lydia - We actually don't know a lot about that. There's a number of studies that have been conducted, looking primarily at the strains that Ian talked about, the predominant strains in Sydney that people may have heard of recently which was last year's strain was reported on quite widely. But one of the important factors is that we only look where people are symptomatic. So, where people are part of an outbreak, those are who are collected into studies. What our study will look at is not only those people, but all the people around them because we're concerned that people are becoming infected and shedding without actually being symptomatic.
Kat - So, the main way that you get rid of virus from your system is by vomiting and diarrhoea. How could people just shed the virus when they're not having severe symptoms?
Lydia - So, the thing about infectious diseases that we're learning is that actually, we have what we call carriage and we have what we call infection or symptoms. And so, you don't actually get rid of the virus by vomiting or having the diarrhoea as Ian had mentioned. You get rid of it by an immune response. And so, the process of shedding the virus happens whilst it's in your body and different people have different immune responses.
Kat - So effectively, you can be not vomiting, not having diarrhoea everywhere, but you can still be dispensing virus to your family, your friends, your colleagues?
Lydia - Exactly, secretly dispensing virus if you will.
Kat - So, what are you trying to do with this study? As you're studying it in people in populations, you're also trying to find out where does it hang out? As Ian said, it can hang around in the environment for up to 10 days.
Lydia - Exactly. So, we're using hospitals as sort of our microcosmic environment if you will and we are swabbing the ward areas. So, people have done this after there's an outbreak and they see lots and lots of norovirus. We want to swab all year round, so we're doing weekly swabbing to see when it's there, if it's there, where it's hiding out, and also, looking at the symptomatic and asymptomatic people.
Kat - And do you have any idea of the kind of patterns that you're looking for?
Lydia - So, what we're looking for and we might be hoping to see is that actually, environmental swabbing will tell us when we should be expecting outbreaks in the hospital. So in other words, we might not see any norovirus, or we might see it at a very low level, and then as that level increases, we wonder if that will lead to outbreaks or not.
Kat - The results from that could be really powerful for hospitals trying to cut down on infections. It surprises me that this kind of thing isn't done already?
Lydia - I would agree with that. I think that there's been a lot of challenges to funding environmental studies and this was the one question I think reviewers had on our study as well. They said, "Well, I'm not sure how strong of a component the environment is, but we like the rest of the study, so go ahead and fund it." But actually, I think the environment is very important. We talk about it a lot for MRSA and other infections that we know about and we just don't know how it's playing a role.
Kat - And it seems quite sobering as well that as Chris said, the advice is, once you've had norovirus, after about 48 hours, you're feeling okay again. It's quite sobering to think that people could be infectious for much longer than that. Do you think that that should change advice on what people should do?
Lydia - At the moment, I would say no. So, we do know from previous studies where they infect people, deliberately, healthy volunteers, with norovirus that the average healthy individual sheds for up to 3 weeks. What we don't know is, how infectious that shedding is and how that contributes to the environment or further outbreaks. So, at the moment, we should probably stick to the guidelines because we don't want healthcare workers certainly off for 3 weeks. That would cause a huge problem for the NHS.
Kat - Well, especially if people are feeling fine again after 2 days, I guess. But thanks very much. That's Lydia Drumwright from Imperial College London.
How norovirus gets inside cells: New clues
Norovirus is the most common viral cause of diarrhea worldwide, but scientists still know little about how it infects people and causes disease. Research has been hindered by an inability to grow the virus in the lab.
Now, researchers at Washington University School of Medicine in St. Louis have identified the protein that norovirus uses to invade cells. The discovery, in mice, provides new ways to study a virus notoriously hard to work with and may lead to treatments or a vaccine.
"Our inability to grow the virus in the lab has limited our ability to develop anti-viral agents. If you can't get the virus to multiply in human cells, how are you going to find compounds that inhibit multiplication?" said Herbert "Skip" Virgin, MD, PhD, the Mallinckrodt Professor and Chair of the Department of Pathology and Immunology and the study's senior author. "This discovery provides a good basis for our mouse model, which we can then use to understand noroviral pathogenesis and search for treatments in people."
The research is published August 18 in Science.
Norovirus is infamous for causing outbreaks of diarrhea, vomiting and stomach cramps on cruise ships, in military barracks and in other environments where people live in close quarters. For most people, infection leads to an uncomfortable day or two punctuated with frequent trips to the bathroom, but in vulnerable populations such as cancer patients and older people, the disease can be long-lasting and sometimes deadly.
There are many noroviruses, but each is restricted to infecting just one animal species. Human norovirus will not infect any of the species typically used in biomedical research, such as mice, rats or rabbits. Human norovirus won't grow even in human cells in petri dishes.
"Since human norovirus won't grow in human cell lines or laboratory animals, you can't test a drug, you can't test a vaccine," Virgin said. "You'd have to do those kinds of studies in people, but it would be better if we can first conduct tests in animal models."
When mouse norovirus was discovered in 2003, it seemed like a great opportunity to make a mouse model of norovirus infection. The genomes of mouse and human norovirus are very similar, and the viruses even look alike under the electron microscope. Nobody could ever be sure, however, that how mouse norovirus acts in mice is relevant to how human norovirus acts in humans.
Virgin and postdoctoral researchers Craig Wilen, MD, PhD, and Robert Orchard, PhD, thought that if they could identify the reason that mouse norovirus infects only mice and human norovirus infects only humans, they could improve their model of norovirus infection.
The researchers used a genetic tool known as CRISPR-Cas9 to identify mouse genes that are important for mouse noroviral infection. They found that when a gene called CD300lf was knocked down by CRISPR-Cas9, norovirus could not infect the cells. CD300lf codes for a protein on the surface of mouse cells, and the researchers believe the virus latches on to it to get inside the cell.
Furthermore, when the researchers expressed mouse CD300lf protein on the surface of human cells, mouse norovirus was able to infect the human cells and multiply. "Mouse norovirus grew just fine in human cells," Virgin said. "This tells us that the species restriction is due to the ability to get inside the cells in the first place. Once inside the cells, most likely all the other mechanisms are conserved between human and mouse noroviruses, since the viruses are so similar."
The researchers also found that mouse norovirus requires a second molecule, or cofactor, to infect cells CD300lf by itself isn't enough. But they were unable to nail down the molecule's identity.
"At this point we know more about what it isn't than what it is," said Orchard, a co-lead author on the study. "Every week there's a new favorite hypothesis. It's probably a small molecule found in the blood, not a protein."
It is unusual for a virus to require a cofactor for infection. Their discovery suggests that the lack of a necessary cofactor may be why scientists have had a difficult time growing human norovirus in the lab.
The researchers are working on ways to use human cells with the mouse CD300lf protein to study noroviral infection. One possibility is to use the system to screen drugs to block viral multiplication. Such drugs could be administered prophylactically to people around the epicenter of an outbreak, or as a treatment for immunocompromised individuals.
The discovery of the mouse receptor for norovirus also could lead to a better understanding of how the virus causes disease.
"We still don't even know if the virus infects epithelial cells or immune cells, and that matters if you want to develop a vaccine," said Wilen, a co-lead author on the study. "We have developed a knockout mouse that lacks CD300lf, and we are using it to identify the cell types involved. We're hoping that a better understanding of the pathogenesis will lead to better ways to treat or prevent this very common disease."